Deep ultraviolet-excitable water-solvated polymeric dyes

ABSTRACT

Water solvated polymeric dyes having a deep ultraviolet excitation spectrum are provided. The subject polymeric dyes include a light harvesting multichromophore having conjugation-modifying repeat units incorporated into the polymer backbone to provide segments of π-conjugated co-monomers having limited π-conjugation between segments. Polymeric tandem dyes are also provided that further include a signaling chromophore covalently linked to the multichromophore in energy-receiving proximity therewith. Also provided are labelled specific binding members that include the subject water solvated polymeric dyes. Methods of evaluating a sample for the presence of a target analyte and methods of labelling a target molecule in which the subject polymeric dyes find use are also provided. Systems and kits for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 62/610,478,filed Dec. 26, 2017, the disclosure of which application is incorporatedherein by reference.

INTRODUCTION

Fluorescent dyes are compounds which, when irradiated with light of awavelength which they absorb, emit light of a (usually) differentwavelength. Fluorescent dyes find use in a variety of applications inbiochemistry, biology and medicine, e.g. in diagnostic kits, inmicroscopy or in drug screening. Fluorescent dyes are characterized by anumber of parameters allowing a user to select a suitable dye dependingon the desired purpose. Parameters of interest include the excitationwavelength maximum, the emission wavelength maximum, the Stokes shift,the extinction coefficient, the fluorescence quantum yield and thefluorescence lifetime. Dyes may be selected according to the applicationof interest in order to, e.g., allow penetration of exciting radiationinto biological samples, to minimize background fluorescence and/or toachieve a high signal-to-noise ratio.

Molecular recognition involves the specific binding of two molecules.Molecules which have binding specificity for a target biomolecule finduse in a variety of research and diagnostic applications, such as thelabelling and separation of analytes, flow cytometry, in situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separations and chromatography. Targetbiomolecules may be detected by labelling with a fluorescent dye.

SUMMARY

Water solvated polymeric dyes having a deep ultraviolet excitationspectrum are provided. The subject polymeric dyes include a lightharvesting multichromophore having conjugation-modifying repeat unitsincorporated into the polymer backbone to provide segments ofπ-conjugated co-monomers having limited π-conjugation between segments.Polymeric tandem dyes are also provided that further include a signalingchromophore covalently linked to the multichromophore inenergy-receiving proximity therewith. Also provided are labelledspecific binding members that include the subject water solvatedpolymeric dyes. Methods of evaluating a sample for the presence of atarget analyte and methods of labelling a target molecule in which thesubject polymeric dyes find use are also provided. Systems and kits forpracticing the subject methods are also provided.

BRIEF DESCRIPTION OF THE FIGURES

It is understood that the drawings, described below, are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 depicts exemplary co-monomers, conjugation-modifying repeat unitsand polymeric dyes of the present disclosure.

FIG. 2 depicts an exemplary pi-conjugated segment of an exemplarypolymeric dye of the present disclosure.

FIG. 3 depicts exemplary polymeric dyes of the present disclosure.

FIG. 4 depicts an exemplary polymeric dye of the present disclosure.

FIG. 5 depicts an exemplary polymeric dye of the present disclosure thatincludes a branched π-conjugated segment.

FIG. 6A-6C depict exemplary polymeric dyes of the present disclosure.

FIG. 7 shows the normalized absorption spectra of several exemplarypolymeric dyes having deep ultraviolet excitation spectrum andabsorption maxima of 350 nm or less.

FIG. 8 shows the normalized emission spectra of several exemplarypolymeric dyes of the present disclosure.

FIG. 9 shows the normalized emission spectra of several exemplarypolymeric tandem dyes having the polymeric dye structure of Dye 1 asdepicted in FIG. 3 conjugated to various signaling chromophores (linkedDye).

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Still, certain terms aredefined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a dye” refersto one or more dyes, i.e., a single dye and multiple dyes. It is furthernoted that the claims can be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

As used herein, the term “sample” relates to a material or mixture ofmaterials, in some cases in liquid form, containing one or more analytesof interest. In some embodiments, the term as used in its broadestsense, refers to any plant, animal or bacterial material containingcells or producing cellular metabolites, such as, for example, tissue orfluid isolated from an individual (including without limitation plasma,serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents, as well as samples from theenvironment. The term “sample” may also refer to a “biological sample”.As used herein, the term “a biological sample” refers to a wholeorganism or a subset of its tissues, cells or component parts (e.g. bodyfluids, including, but not limited to, blood, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen). A “biological sample” canalso refer to a homogenate, lysate or extract prepared from a wholeorganism or a subset of its tissues, cells or component parts, or afraction or portion thereof, including but not limited to, plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors and organs. In certain embodiments, the sample hasbeen removed from an animal or plant. Biological samples may includecells. The term “cells” is used in its conventional sense to refer tothe basic structural unit of living organisms, both eukaryotic andprokaryotic, having at least a nucleus and a cell membrane. In certainembodiments, cells include prokaryotic cells, such as from bacteria. Inother embodiments, cells include eukaryotic cells, such as cellsobtained from biological samples from animals, plants or fungi.

As used herein, the terms “support bound” and “linked to a support” areused interchangeably and refer to a moiety (e.g., a specific bindingmember) that is linked covalently or non-covalently to a support ofinterest. Covalent linking may involve the chemical reaction of twocompatible functional groups (e.g., two chemoselective functionalgroups, an electrophile and a nucleophile, etc.) to form a covalent bondbetween the two moieties of interest (e.g. a support and a specificbinding member). In some cases, non-covalent linking may involvespecific binding between two moieties of interest (e.g., two affinitymoieties such as a hapten and an antibody or a biotin moiety and astreptavidin, etc.). In certain cases, non-covalent linking may involveabsorption to a substrate.

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length,e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length.

As used herein the term “isolated,” refers to an moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 5 or more, such as 10 or more, 100 or more, 1000 or more,10,000 or more, 100,000 or more, 10⁶ or more, 10⁷ or more, 10⁸ or moreor 10⁹ or more members.

Numeric ranges are inclusive of the numbers defining the range.

As used herein, the term “specific binding” refers to the ability of acapture agent (or a first member of a specific binding pair) topreferentially bind to a particular analyte (or a second member of aspecific binding pair) that is present, e.g., in a homogeneous mixtureof different analytes. In some instances, a specific binding interactionwill discriminate between desirable and undesirable analytes in a samplewith a specificity of 10-fold or more for a desirable analyte over anundesirable analytes, such as 100-fold or more, or 1000-fold or more. Insome cases, the affinity between a capture agent and analyte when theyare specifically bound in a capture agent/analyte complex is at least10⁻⁸ M, at least 10⁻⁹M, such as up to 10⁻¹⁰ M.

As used herein, the terms “affinity” and “avidity” have the same meaningand may be used interchangeably herein. “Affinity” refers to thestrength of binding, increased binding affinity being correlated with alower Kd.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

As used herein, the terms “evaluating”, “determining,” “measuring,” and“assessing,” and “assaying” are used interchangeably and include bothquantitative and qualitative determinations.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

As used herein, the term “linker” or “linkage” refers to a linkingmoiety that connects two groups and has a backbone of 100 atoms or lessin length. A linker or linkage may be a covalent bond that connects twogroups or a chain of between 1 and 100 atoms in length, for example achain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbonatoms in length, where the linker may be linear, branched, cyclic or asingle atom. In some cases, the linker is a branching linker that refersto a linking moiety that connects three or more groups. In certaincases, one, two, three, four or five or more carbon atoms of a linkerbackbone may be optionally substituted with a sulfur, nitrogen or oxygenheteroatom. In some cases, the linker backbone includes a linkingfunctional group, such as an ether, thioether, amino, amide,sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioesteror imine. The bonds between backbone atoms may be saturated orunsaturated, and in some cases not more than one, two, or threeunsaturated bonds are present in a linker backbone. The linker mayinclude one or more substituent groups, for example with an alkyl, arylor alkenyl group. A linker may include, without limitations,polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, whichmay be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable.

As used herein, the terms “polyethylene oxide”, “PEO”, “polyethyleneglycol” and “PEG” are used interchangeably and refer to a polymericgroup including a chain described by the formula —(CH₂—CH₂—O—)_(n)— or aderivative thereof. In some embodiments, “n” is 5000 or less, such as1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 orless, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10 to 15.It is understood that the PEG polymeric group may be of any convenientlength and may include a variety of terminal groups and/or furthersubstituent groups, including but not limited to, alkyl, aryl, hydroxyl,amino, acyl, acyloxy, and amido terminal and/or substituent groups. PEGgroups that may be adapted for use in the subject multichromophoresinclude those PEGs described by S. Zalipsky in “Functionalizedpoly(ethylene glycol) for preparation of biologically relevantconjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165; and by Zhu etal in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, andTherapy”, Chem. Rev., 2012, 112 (8), pp 4687-4735.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Alkyl groups of interest include,but are not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group includes from 1 to 20 carbon atoms. In some embodiments, analkyl group includes from 1 to 10 carbon atoms. In certain embodiments,an alkyl group includes from 1 to 6 carbon atoms, such as from 1 to 4carbon atoms. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—),n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Amino” refers to the group —NH₂. The term “substituted amino” refers tothe group —NRR where each R is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl,heteroaryl, and heterocyclyl provided that at least one R is nothydrogen.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system. Arylgroups of interest include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupincludes from 6 to 20 carbon atoms. In certain embodiments, an arylgroup includes from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Substituted aryl”, unless otherwise constrained by the definition forthe aryl substituent, refers to an aryl group substituted with from 1 to5 substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl and trihalomethyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Heteroarylgroups of interest include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene,triazole, xanthene, benzodioxole and the like. In certain embodiments,the heteroaryl group is from 5-20 membered heteroaryl. In certainembodiments, the heteroaryl group is from 5-10 membered heteroaryl. Incertain embodiments, heteroaryl groups are those derived from thiophene,pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline,imidazole, oxazole and pyrazine.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

“Substituted heteroaryl”, unless otherwise constrained by the definitionfor the substituent, refers to an heteroaryl group substituted with from1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl and trihalomethyl.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl andsubstituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Substituents of interest include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰) (O⁻),—OP(O)(OR⁶⁰) (OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O⁻, —C(S) OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Forexample, a substituted group may bear a methylenedioxy substituent orone, two, or three substituents selected from a halogen atom, a(1-4C)alkyl group and a (1-4C)alkoxy group. When the group beingsubstituted is an aryl or heteroaryl group, the substituent(s) (e.g., asdescribed herein) may be referred to as “aryl substituent(s)”.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

As summarized above, water solvated polymeric dyes having a deepultraviolet excitation spectrum are provided. The subject polymeric dyesinclude a light harvesting multichromophore having conjugation-modifyingrepeat units incorporated into the polymer backbone to provide segmentsof π-conjugated co-monomers having limited π-conjugation betweensegments. In some cases, the polymeric dye includes a plurality ofπ-conjugated segments separated by conjugation-modifying repeat units.Polymeric tandem dyes are also provided that further include a signalingchromophore covalently linked to the polymeric dye in energy-receivingproximity therewith. Also provided are labelled specific binding membersthat include the subject water solvated polymeric dyes. Methods ofevaluating a sample for the presence of a target analyte and methods oflabelling a target molecule in which the subject polymeric dyes find useare also provided. Systems and kits for practicing the subject methodsare also provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

In further describing the subject invention, water solvated polymericdyes including light harvesting multichromophores and related polymerictandem dyes further including a signaling chromophore are describedfirst in greater detail. Next, labelled specific binding members whichinclude the subject polymeric dyes are described. Then, methods ofinterest in which the subject polymeric dyes find use are reviewed.Systems and kits that may be used in practicing methods of the presentdisclosure are also described.

Polymeric Dyes

As summarized above, the present disclosure provides water solvatedpolymeric dyes having a deep ultraviolet excitation spectrum and/or adeep UV absorption maximum. Deep ultraviolet (Deep UV or DUV) is aregion of the electromagnetic spectrum from about 200 nm to about 350nm, such as from 250 nm to 350 nm. Deep UV includes ultraviolet B (UVB)and ultraviolet C (UVC) radiation, where UVB radiation has wavelengthsin the range of 290 to 320 nm and UVC radiation has wavelengths in therange of 200 nm to 290 nm.

The term “deep ultraviolet excitation spectrum” can refer to theabsorption spectrum of a polymeric dye that has a full width at halfmaximum (FWHM) defining a wavelength range that is inside the deep UVregion of the electromagnetic spectrum. As such, the absorption profileof such a polymeric dye is located in the deep UV region. FIG. 7 showsthe normalized absorption spectra of several exemplary polymeric dyeshaving FWHM defining wavelengths in the deep UV. The subject polymericdyes can absorb strongly at wavelengths in the range from 250 nm to 350nm.

The term “deep UV absorption maximum” refers to an absorption maximumwavelength of a polymeric dye that is in the deep UV region of theelectromagnetic spectrum, e.g., an absorption maximum wavelength of 350nm or less, such as an absorption maximum wavelength in the range of 200nm to 350 nm, e.g., 200 to 250 nm, 250 nm to 290 nm, 290 to 320 nm or320 nm to 350 nm. In some embodiments, a “deep UV absorption maximum” isan absorption maximum wavelength of 340 nm or less, such as 330 nm orless, 320 nm or less, 310 nm or less, 300 nm or less, 290 nm or less,280 nm or less, 270 nm or less, or even less. FIG. 7 shows thenormalized absorption spectra of several exemplary polymeric dyes havingdeep UV absorption maxima of 350 nm or less and deep ultravioletexcitation spectra have FWHM in the range of 200 nm to 350 nm.

The subject water solvated polymeric dyes find use in a variety ofmultiplexed applications where a panel of fluorescent dyes can beindependently excited to provide emissions for high parameter analyses.The polymeric dyes can be used directly as fluorescent labels or coupledwith a small molecule acceptor signaling chromophore in a tandem dye toprovide for a variety of emission wavelengths over a range of 320 to1200 nm. In multi-color applications, use multiple excitation sourcescan achieving multiplicity and a high number of parameters. In additionto narrow peak width, minimizing absorption at other common excitationsources is important for higher parameter experiments. The degree ofcross-source excitation can be tuned for the subject polymeric dyes byselection of a particular structure, co-monomers, and mixtures ofco-blocks.

The subject polymeric dyes include a light harvesting multichromophorehaving segment(s) of π-conjugated co-monomers and conjugation-modifyingrepeat unit(s). The light harvesting multichromophore of the subjectpolymeric dyes can provide a desirable absorption profile in the deep UVrange without significant absorption at longer wavelengths. A desireddeep UV absorption profile can be accomplished by incorporatingconjugation-modifying repeat units in the main chain (or backbone) of apolymeric dye, to connect adjacent pi-conjugated segments via covalentbond(s) which units can prevent or reduce the degree of p-orbitaloverlap (and thus limit the π-conjugation) across these covalentbond(s). The conjugation-modifying repeat units are capable ofmodulating (e.g., reducing) the extended pi-conjugation that wouldotherwise be present along a polymeric dye backbone of conjugatedco-monomers lacking such repeat units. Without suchconjugation-modifying repeat units, a polymeric dye composed solely ofthe remaining pi-conjugated co-monomers would have extended electronicdelocalization and narrow band gaps. The conjugation-modifying repeatunits of the subject polymeric dyes directly connect and modulateconjugation between adjacent segments of pi-conjugated co-monomers toprovide a desirable effective conjugation length. By limiting theeffective conjugation length of the resulting light harvestingmultichromophore, wider electronic band gaps can be achieved to providefor an absorption profile including short wavelengths of light in thedeep UV range. As such, the subject polymeric dyes expand the spectralspace into the deep UV over spectral coverage provided by conventionaldyes and instruments.

Conjugation Modifying Repeat Units

As used herein, the term “conjugation modifying repeat unit” refers to adivalent unit of the polymer backbone that is connected to adjacentsegments of pi-conjugated co-monomers and which includes, within theunit, linking covalent bond(s) that prevent or reduce p-orbital overlapacross the covalent bond(s) thereby limiting electronic delocalizationbetween the two adjacent pi-conjugated segments. In some cases, thelinking covalent bonds can be defined by a divalent atom, group orlinker that is directly covalently linked to adjacent units of thepolymer backbone. In certain cases, the linking covalent bond is definedby a conjugation modifying repeat unit including two unsaturated groups(e.g., aryl, heteroaryl or alkenyl groups) that are separated in therepeat unit by a sterically strained single covalent bond.

The conjugation-modifying repeat unit can limit electron delocalizationalong the polymer backbone and thus limit the effective conjugationlength. In some cases, the conjugation-modifying repeat unitincorporates into the backbone of the polymeric dye, an atom, functionalgroup or linker (Z) that provides saturated or partially saturatedcovalent bonds which limit or prevent pi-conjugation across thosecovalent bonds. In certain cases, the conjugation-modifying repeat unitincludes a strained single covalent bond (Z) linking two aryl orheteroaryl groups but limiting p-orbital overlap across the bond. Thesingle covalent bond can be strained by torsion created by incorporationof adjacent sterically hindered substituents on the aryl or heteroarylthat twist the two aryl or heteroaryl groups out of planarity.

The subject polymeric dyes can include a plurality of π-conjugatedsegments of co-monomers connected via conjugation modifying repeatunits. The π-conjugated segments can include a single co-monomer, two ormore co-monomers or a larger plurality of co-monomers. In someinstances, the π-conjugated segments have an effective conjugationlength of 30 or less, such as 20 or less, 18 or less, 16 or less, 14 orless, 12 or less, 10 or less, 8 or less, 6 or less, or 5 or less. Anyconvenient arrangements of π-conjugated segments and conjugationmodifying repeat units can be utilized to provide for a desiredeffective conjugation length. In some cases, the polymeric dye is analternating co-polymer of particular π-conjugated segments andconjugation modifying repeat units. In certain cases, the polymeric dyeis a random co-polymer of co-monomers where the conjugation modifyingrepeat units are incorporated into the polymer with a frequency or %molarity to give an average π-conjugated segment size that provides adesired effective conjugation length in the polymer.

A conjugation-modifying repeat unit can comprise two aryl or heteroarylgroups covalently linked to each other via a conjugation-modifying atom,group or linker (Z) that prevents or limits conjugation between the twoaryl or heteroaryl groups, e.g., a unit of the formula —Ar¹—Z—Ar²—wherein Ar¹ and Ar² are the two aryl or heteroaryl groups and Z is theconjugation-modifying atom, group or linker (Z). The two aryl orheteroaryl groups (e.g., Ar¹ and Ar²) can be 1,4-linked or 1,3-linked tothe polymeric dye backbone, and can be unsubstituted or furthersubstituted, e.g., with a water solubilizing group (e.g., as describedherein). The two aryl or heteroaryl groups (e.g., Ar¹ and Ar²) can bemonocyclic or bicyclic. In some cases, the two aryl or heteroaryl groups(e.g., Ar¹ and Ar²) are independently selected from 1,4-phenyl,1,3-phenyl, 2,5-pyridyl, 2,6-pyridyl and 3,5-pyridyl, optionally furthersubstituted. In certain cases, the conjugation-modifying repeat unit hasa formula —Ar¹—Z—Ar²— wherein Ar¹ and Ar² are each independently a1,4-phenyl or a 2,5-pyridyl, optionally further substituted. In someinstances, a conjugation-modifying repeat unit has a structure offormula (XI):

wherein:

Z is selected from a covalent bond, a saturated atom, a saturated groupand a functional group, with the proviso that when Z is a covalent bondat least two (e.g., 2, 3 or 4) of R²², R²³, R²⁵ and R²⁸ are not H;

R²⁸ are independently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide, and a water solubilizing group,and wherein one or more of R²², R²³, R²⁵ and R²⁸ is optionallycyclically linked to Z to provide a benzo-fused carbocycle orheterocycle ring; and

each * is a site for covalent attachment to the backbone of thepolymeric dye. In some instances of formula (XI), when Z is a covalentbond all of R²², R²³, R²⁵ and R²⁸ are not H, such that there is reducedp-orbital overlap across Z due to steric strain. As such, z can be asterically strained single covalent bond in a conjugation-modifyingrepeat unit of formula (XI).

In certain embodiments of formula (XI), Z is selected from a saturatedcarbon atom, a saturated heteroatom, a functional group and a saturatedlinker. A saturated carbon atom (Z) of formula (XI) can be covalentlybonded to the adjacent phenyl groups via two single saturated covalentbonds, thereby preventing or limiting orbital overlap between the twophenyl rings. In certain instances, Z is C(R⁵)(R⁶)— wherein R⁵ and R⁶are independently H, alkyl or substituted alkyl. Similarly, a saturatedheteroatom (Z) of formula (XI) refers to a heteroatom, such as oxygen,sulfur, nitrogen or silicon that is covalently bonded to the adjacentphenyl groups via two single saturated covalent bonds, therebypreventing or limiting orbital overlap between the two phenyl rings. Incertain instances, Z is O, S or NR′ or —Si(R⁵)(R⁶)— wherein R′, R⁵ andR⁶ are independently H, alkyl or substituted alkyl. Similarly, asaturated linker can provide a backbone of three or more saturatedcovalent bonds between the two adjacent phenyl rings of formula (XI).Any convenient linkers can find use in the repeat unit of formula (XI).Linkers of interest that find use as Z in formula (XI) include, but arenot limited to, alkyl linkers, such as a C₂-C₁₂ substituted orunsubstituted alkyl linker or a C₂-C₆ substituted or unsubstituted alkyllinker, a polyethylene glycol linker (e.g., —O—(CH₂CH₂O)_(n)— where n is1-6, such as 1, 2, 3, 4, 5 or 6) or a modified polyethylene glycol (PEG)linker (e.g., a substituted PEG, or a terminal modified PEG, e.g.,—Z—(CH₂CH₂O)_(n)—).

A functional group (Z) of formula (XI) can refer to aheteroatom-containing group of atoms having characteristic properties,which provides a linkage between the two phenyl groups of formula (XI)but that prevents or limits conjugation between the phenyl groups.Functional groups of interest that find use in conjugation modifyingrepeat units include, but are not limited to, oxygen, sulfur and/ornitrogen containing functional groups, such as a sulfonyl, a keto, anamide, ester, carbonate, carbamate or urea. Exemplary functional groupsinclude —S(O)—, —SO₂—, —SO₂NR′—, —S(O)O—, —C(O)—, —C(O)NR′—, —C(O)O—,—OC(O)NR′—, —OC(O)O—, —NR′C(O)NR′—, where R′ is H, alkyl or substitutedalkyl. Exemplary conjugation-modifying repeat units of interest include,but are not limited to:

In some embodiments of formula (XI), Z is a covalent bond, at least oneof R²² and R²³ is not H and at least one of R²⁵ and R²⁸ is not H. Insome cases, three of R²², R²³, R²⁵ and R²⁸ are not H. In some instances,all of R²², R²³, R²⁵ and R²⁸ are not H. In some instances, two or moreof R²², R²³, R²⁵ and R²⁸ are water solubilizing groups. In certaininstances, R²³ and R²⁵ are each H. In such cases, the Z bond of theconjugation-modifying repeat unit is torsionally strained by theselection of substituents at R²², R²³, R²⁵ and/or R²⁸ thereby reduceplanarity and orbital overlap between the two adjacent phenyl rings. Insuch cases, the conjugation-modifying repeat unit can modulate (e.g.,limit) electronic delocalization between flanking π-conjugated segmentsor co-monomers to which the two 1,4-phenyl rings of formula (XI) areconjugated.

In some embodiments of formula (XI), Z is a covalent bond and theconjugation-modifying repeat unit comprises the structure:

wherein:

R¹ to R⁴ are independently selected from hydrogen, alkyl, substitutedalkyl, halogen (e.g., fluoro), cyano,

wherein X is O, S, CONH or NH, with the proviso that R¹ and R² are nothydrogen; each R⁵ is independently —(CH₂CH₂O)_(p)R′ wherein p is aninteger from 0 to 50 and R′ is H, an alkyl or a substituted alkyl; and mis an integer from 0 to 10. In certain instances of formula (XIII), atleast two of R¹ to R⁴ are selected from:

wherein X is O, S, CONH or NH, wherein each R⁵ is independently—(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50 and R′ is H, analkyl or a substituted alkyl. In certain instances of formula (XIII), R¹and R² are selected from:

wherein X is O, S, CONH or NH, wherein each R⁵ is independently—(CH₂CH₂O)_(p)R′ wherein each p is independently an integer from 0 to 50(such as 6-50, 6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20) and R′ is H, an alkyl or a substitutedalkyl.

Exemplary conjugation-modifying repeat units of interest include, butare not limited to:

each R⁵ is independently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0to 50 (e.g., 0 to 30, 0 to 20, or 10 to 20) and R′ is H, a lower alkylor a substituted lower alkyl. In certain instances ofconjugation-modifying repeat units, in a R⁵ water soluble polymer, p is7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and R′ is methyl.In certain cases, each p is 10, 11 or 12.

In some instances of formula (XI), Z is C(R⁵)(R⁶)— wherein R⁵ and R⁶ areeach cyclically linked to an adjacent phenyl ring of formula (XI), e.g.,cyclically linked to the R²² or R²³ position, or cyclically linked tothe R²⁵ or R²⁸ provide a benzo-fused carbocycle or heterocycle ring. Incertain instances of formula (XI), Z is —C(R⁵)(R⁶)—, wherein R⁵ iscyclically linked to R²² to provide a partially saturated carbocycle(e.g., 5 or 6 membered carbocycle) that is optionally furthersubstituted, and R⁶ is cyclically linked to R²⁸ provide a partiallysaturated carbocycle (e.g., 5 or 6 membered carbocycle) that isoptionally further substituted. In certain embodiments of formula (XI),the conjugation-modifying repeat unit has a structure of formula (XII):

wherein:

n¹, n², m¹ and m² are independently 0-3, wherein n¹+m¹ and n²+m² areeach independently 2-3;

R¹⁶ and R¹⁷ are each independently selected from H, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g.,fluoro, chloro or bromo), sulfonate, sulfonamide, substitutedsulfonamide, carboxy, carboxyamide, substituted carboxyamide and a watersolubilizing group; and

each * is a site for covalent attachment to the backbone of thepolymeric dye. In certain instances of formula (XII), n¹+m¹ and n²+m²are each 2. In certain instances of formula (XII), n¹+m¹ and n²+m² areeach 3. In certain instances of formula (XII), each R¹⁶ and R¹⁷ are H.In certain instances of formula (XII), m² and m² are each 1, and atleast one of R¹⁶ and R¹⁷ is not H. In certain instances of formula(XII), m² and m² are each 1, and at least one or both of R¹⁶ and R¹⁷ isa water solubilizing group.

Exemplary conjugation-modifying repeat units of interest include, butare not limited to:

As used herein, the terms “light harvesting multichromophore”,“polymeric dye” and “conjugated polymer” are used interchangeably andrefer to a conjugated polymer which has a structure capable ofharvesting light with a particular absorption maximum wavelength andconverting it to emitted light at a longer emission maximum wavelength.In some cases, the light harvesting multichromophore is itselffluorescent. Conjugated polymers (CPs) are characterized by adelocalized electronic structure and may have an effective conjugationlength that is substantially shorter than the length of the polymerchain, because the backbone may contain a large number of conjugatedsegments in close proximity. In some cases, conjugated polymers areefficient for light harvesting and provide for optical amplification viaForster energy transfer to an acceptor.

As used herein the term “unit” refers to a structural subunit of apolymer. The term unit is meant to include monomers, co-monomers,co-blocks, conjugated segments, repeating units, and the like. A“repeating unit” is a subunit of a polymer that is defined by theminimum number of distinct structural features that are required for theunit to be considered monomeric, such that when the unit is repeated ntimes, the resulting structure describes the polymer or a block thereof.In some cases, the polymer may include two or more different repeatingunits, e.g., when the polymer is a multiblock polymer or a randomarrangement of units, each block may define a distinct repeating unit,e.g., an n-block and a m-block. It is understood that a variety ofarrangements of n and/or m repeating units or blocks are possible andthat in the depicted formula of the subject multichromophores describedherein any convenient linear arrangements of n and m co-blocks ofvarious lengths are included within the structure of the overallpolymer. It is understood that the polymer may also be represented by aformula in terms of mol % values of each unit in the polymer and thatsuch formula may represent a variety of arrangements of repeat unit,such as random or multiblock polymer. In some cases, a repeating unit ofthe polymer includes a single monomer group. In certain instances, arepeating unit of the polymer includes two or more monomer groups, i.e.,co-monomer groups, such as two, three, four or more co-monomer groups.As used herein, the term “co-monomer” or “co-monomer group” refers to astructural unit of a polymer that may itself be part of a repeating unitof the polymer. In some embodiments, the conjugated polymer includes ablock copolymer that is composed of blocks of polymerized monomers. Insuch cases, the block copolymer may be described as having distinctrepeating units each corresponding to a distinct co-block of thepolymer. In some cases, the polymer is a diblock copolymer that containstwo different co-blocks. In such cases, the polymer may be described asincluding co-blocks, where each co-block may be composed of co-monomers,such as one, two, three or more co-monomers.

The multichromophore may have any convenient length. In some cases, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 500,000, such as 2 to100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units orsegments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to100,000, or 500 to 50,000 units or segments. In some instances, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 1,000, such as 2 to500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to100, 9 to 100 or 10 to 100 units or segments.

The multichromophore may be of any convenient molecular weight (MW). Insome cases, the MW of the multichromophore may be expressed as anaverage molecular weight. In some instances, the polymeric dye has anaverage molecular weight in the range of 500 to 500,000, such as from1,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000 or evenan average molecular weight in the range of 50,000 to 100,000.

In some embodiments, the multichromophore includes a particularco-monomer (e.g., an aryl or heteroaryl co-monomer) that constitutes 5%or more by molarity (e.g., 5 mol %) of the multichromophore, such as 10%or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% ormore, 45% or more, 50% or more, 60% or more, 70% or more, or even moreby molarity of the multichromophore. In such cases, the multichromophoremay include 5 or more repeating units, such as 10 or more, 20 or more,30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more,90 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 10,000or more, or even more repeating units. In such cases, themultichromophore may include 5 or more co-monomer units, such as 10 ormore, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 ormore, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more,1000 or more, 10,000 or more, or even more co-monomer units. In certainembodiments, a co-monomer (e.g., aryl or heteroaryl co-monomer) ofinterest constitutes 25% or more by molarity of the multichromophore,such as 30% or more, 40% or more, 45% or more, 50% or more, or even moreby molarity of the multichromophore, which includes 5 or more repeatingunits, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 ormore, 60 or more, 70 or more, 80 or more, 90 or more, 100 or morerepeating units.

In some embodiments, the multichromophore includes a plurality of firstoptically active units forming a conjugated system or system ofconjugated segments (e.g., as described herein), having an absorptionwavelength (e.g., as described herein) at which the first opticallyactive units absorb light to form an excited state. The subjectmultichromophore may have one or more desirable spectroscopicproperties, such as a particular deep UV absorption profile or deep UVmaximum wavelength (e.g., as described herein), a particular emissionmaximum wavelength, extinction coefficient, quantum yield, and the like.The subject polymeric dyes and polymeric tandem dyes provide for a deepUV absorption spectrum and a variety of emission profiles which dependon a variety of factors such as the selected co-monomers, linkinggroups, substituents and optional linked signaling chromophores of whichthe polymers are composed.

In some embodiments, the multichromophore itself has an emission maximumwavelength in the range of 300 to 900 nm, such as 300 to 850 nm, 300 to600 nm, 300 to 500 nm, 300 to 500 nm, 300 to 500 nm, 350 to 500 nm or400 to 500 nm, where specific examples of emission maxima of interestinclude, but are not limited to: 340 nm±5 nm, 350 nm±5 nm, 360 nm±5 nm,370 nm±5 nm, 380 nm±5 nm, 390 nm±5 nm, 400 nm±5 nm, 410 nm±5 nm, 420nm±5 nm, or 430 nm±5 nm. In certain instances, the multichromophore islinked to an acceptor signaling chromophore, which polymeric tandem dyehas an emission maximum wavelength selected from the group consisting of395 nm±5 nm, 460 nm±5 nm, 490 nm±5 nm, 550 nm±5 nm, 560 nm±5 nm, 605nm±5 nm, 650 nm±5 nm, 680 nm±5 nm, 700 nm±5 nm and 805 nm±5 nm. In someinstances, the polymeric tandem dye has an emission maximum wavelengthin the range of 375 to 900 nm (such as in the range of 380 nm to 900 nm,390 nm to 900 nm, or 400 nm to 900 nm).

In some instances, the multichromophore has an extinction coefficient of5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹ or more, 7×10⁵ cm⁻¹M⁻¹ ormore, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ or more, such as 1×10⁶cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶ _(cm) ⁻¹M⁻¹ or more,2.5×10⁶ cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶ cm⁻¹M⁻¹ or more,5×10⁶ cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶ cm⁻¹M⁻¹ or more, or8×10⁶ cm⁻¹M⁻¹ or more. In such cases, the multichromophore may have 5 ormore repeating units, such as 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, 50 or more, 100 or more, or even more repeating units.In some embodiments, the multichromophore has a molar extinctioncoefficient of 5×10⁵ M⁻¹ cm⁻¹ or more. In certain embodiments, themultichromophore has a molar extinction coefficient of 1×10⁶ M⁻¹ cm⁻¹ ormore.

In some instances, the multichromophore has an extinction coefficient of40,000 cm⁻¹M⁻¹ per repeating unit or more, such as 45,000 cm⁻¹M⁻¹ perrepeating unit or more, 50,000 cm⁻¹M⁻¹ per repeating unit or more,55,000 cm⁻¹M⁻¹ per repeating unit or more, 60,000 cm⁻¹M⁻¹ per repeatingunit or more, 70,000 cm⁻¹M⁻¹ per repeating unit or more, 80,000 cm⁻¹M⁻¹per repeating unit or more, 90,000 cm⁻¹M⁻¹ per repeating unit or more,100,000 cm⁻¹M⁻¹ per repeating unit or more, or even more. In someinstances, the 40,000 cm⁻¹M⁻¹ per repeating unit or more describedherein is an average extinction coefficient. In certain instances, therepeat unit of the multichromophore may include a single monomer, twoco-monomers, or three or more co-monomers. In some instances, themultichromophore has an extinction coefficient of 40,000 cm⁻¹M⁻¹ perco-monomer or more, such as 45,000 cm⁻¹M⁻¹ per co-monomer or more,50,000 cm⁻¹M⁻¹ per co-monomer or more, 55,000 cm⁻¹M⁻¹ per co-monomer ormore, 60,000 cm⁻¹M⁻¹ per co-monomer or more, 70,000 cm⁻¹M⁻¹ perco-monomer or more, 80,000 cm⁻¹M⁻¹ per co-monomer or more, 90,000cm⁻¹M⁻¹ per co-monomer or more, 100,000 cm⁻¹M⁻¹ per co-monomer or more,or even more. In some instances, the 40,000 cm⁻¹M⁻¹ per co-monomer ormore is an average extinction coefficient.

π-Conjugated Segments

As summarized above, the subject polymeric dyes include π-conjugatedsegments whose effective conjugation length are controlled by theincorporation of conjugation-modifying repeat units (e.g., as describedherein) into the polymer. The terms “π-conjugated segment” and “segmentof π-conjugated co-monomers” are used interchangeably herein and referto a region of pi-conjugated co-monomers in the polymeric backbone, oron a conjugated branch of the polymeric backbone. The terms“π-conjugated” and “pi-conjugated” are used interchangeably herein. Anyconvenient arrangements of π-conjugated segments and conjugationmodifying repeat units can be utilized to provide for a desiredeffective conjugation length and resulting spectroscopic properties. Itis understood that, in some cases, the subject multichromophores mayinclude co-blocks (e.g., n and m co-blocks) or a random arrangement ofrepeating units or co-monomers. It is understood that the formula hereindepict a representation of the polymeric dye, and that the chemicalstructure of the polymer may vary in length and/or the particulararrangement of co-monomers, depending on the method of synthesis. Thesubject multichromophores may include any convenient linear arrangementsof n and m co-blocks of various lengths within the structure of theoverall polymer. The subject multichromophores may include anyconvenient linear arrangements of co-monomers of within the structure ofthe overall polymer, depending on the particular synthesis andpolymerization method used. In some cases, the linear arrangements ofco-monomers depicted in the structures herein are random. In addition,the multichromophores may include any convenient arrangements ofco-monomers within such n and/or m co-blocks, including random,alternating or block arrangements of co-monomers. Unless indicated tothe contrary, all possible arrangements of co-monomers are meant to beincluded in the polymeric dyes described herein. A variety of polymersynthesis methods may be utilized to prepare co-monomers and co-blocksof interest in the preparation of the subject multichromophores. It isunderstood that in some cases, the polymerization methods may produce acomposition including a population of conjugated polymers that includessome variation with respect to the particular length and/or terminalgroups (i.e., end groups) present in each conjugated polymer of thepopulation. The formulae depicted herein may refer to a single compoundor to a population or sub-population of polymeric compounds. As usedherein, * denotes a site for covalent attachment to the unsaturatedbackbone of a conjugated polymer. It is understood that the formulaedepicted herein of the subject polymers is one representation of thestructure and that other representations, such as a representationindicating mol % ratios of particular co-monomers in the conjugatedpolymer may also be used.

The π-conjugated segments of the subject polymeric dye can be composedof any convenient co-monomers capable of pi-conjugation, such as aryl orheteroaryl co-monomers, alkynyl co-monomers (e.g., ethynylene) orsegments and alkenyl co-monomers (e.g., vinylene) or segments. Aryl orheteroaryl co-monomers of interest which find use in the π-conjugatedsegments include, but are not limited to, phenyl co-monomers, biphenylco-monomers, benzooxazole co-monomers, benzothiazole co-monomers,poly-phenylene co-monomers, and fused tricyclic co-monomers, such asfluorene co-monomers, carbazole co-monomers, silole co-monomers andbridged biphenyl co-monomers. The aryl or heteroaryl co-monomers may beoptionally further substituted, e.g., as described herein.

Various co-monomers which find use in π-conjugated segments of thesubject polymeric dyes are now described in more detail.

In some embodiments, the polymeric dye comprises a plurality ofπ-conjugated segments each comprising one or more co-monomers selectedfrom the following structures (a)-(k):

wherein:

X is O or S;

n is 1-6 (e.g., 1, 2 or 3);

Y¹ is selected from —C(R¹¹)₂—, —Si(R¹¹)₂—, —C(R¹¹)₂C(R¹¹)₂— and—C(R¹¹)₂C(R¹¹)₂—;

R¹¹ is one or more optional substituents each independently selectedfrom hydrogen, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy,hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group; and

each * is a site for covalent attachment to the backbone of the polymer.

In certain cases of structures (a)-(k), each R¹¹ is independentlyselected from H, F, methyl, trifluoromethyl, cyano,

wherein:

X is O or S;

m is an integer from 0-10; and

R⁵ is —(CH₂CH₂O)_(p)R′ wherein R′ is H, alkyl or substituted alkyl(e.g., methyl) and p is an integer from 0-40.

In some embodiments, the polymeric dye comprises a plurality ofπ-conjugated segments each comprising one or more co-monomers selectedfrom alkynyl co-monomers (e.g., ethynylene) and alkenyl co-monomers(e.g., vinylene) of structures (j)-(k):

where R¹¹ is selected from hydrogen, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo),sulfonate, sulfonamide, substituted sulfonamide, carboxy, carboxyamide,substituted carboxyamide and a water solubilizing group (e.g., asdescribed herein).Aryl or Heteroaryl Co-Monomers

As summarized above, the subject polymeric dyes include conjugatedsegments of aryl or heteroaryl co-monomers linked via covalent bonds,vinylene groups or ethynylene groups. In addition to the co-monomersdescribed in the exemplary formulae above, a variety of aryl andheteroaryl co-monomers find use in the subject polymeric dyes. Anyconvenient aryl and heteroaryl co-monomers may be utilized in thesubject multichromophores, e.g., in a band gap modifying unit, and maybe evenly or randomly distributed along the conjugated polymer asdescribed herein.

In some instances, the aryl or heteroaryl co-monomer is a substituted orunsubstituted phenyl, biphenyl or pyridyl co-monomer. In certainembodiments, the aryl or heteroaryl co-monomer is selected fromsubstituted or unsubstituted 1,4-phenyl, a substituted or unsubstituted1,3-phenyl, a substituted or unsubstituted 4,4′-biphenyl, a substitutedor unsubstituted 2,5-pyridyl, and a substituted or unsubstituted2,6-pyridyl. In certain instances, the co-monomer is an optionallysubstituted aryl or heteroaryl co-monomer selected from one of thefollowing structures:

where Z²-Z⁵ are each independently CR or N, where at least one Z²-Z⁵ isN; and each R and each R¹¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, water solubilizing group, halogen, cyano,alkoxy, substituted alkoxy, alkyl and substituted alkyl. In certainembodiments, one and only one of Z²-Z⁵ is N. In certain embodiments, twoand only two of Z²-Z⁵ is N. In certain instances, R¹¹, R¹² and R¹⁴ areeach H. In some instances, R¹² and R¹⁴ are each H. In some instances,R¹¹ and R¹³ are each H. In some cases, R¹⁵ and R¹⁶ are each H. In someinstances, the halogen is fluoro.

In some cases, the aryl or heteroaryl co-monomer (e.g., structure (a))is selected from one of the following:

wherein R¹¹ and R¹² are independently hydrogen, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide and a water solubilizing group.In certain cases, the aryl or heteroaryl co-monomer selected from one ofthe following:

where n is 1-20 and R′ is H or lower alkyl. In some embodiments of thearyl or heteroaryl co-monomer structures, n is an integer from 6 to 20,such as 12-20 or 12-16 or 16-20.

Also provided are substituted phenylene co-monomers that are substitutedwith a further aryl or heteroaryl group which is itself optionallyfurther substituted. The aryl or heteroaryl-substituted phenyleneco-monomers can provide for extended pi-conjugation into a sidechaingroup of the co-monomer. In certain cases, the co-monomer is aheteroaryl-substituted phenylene co-monomer. Selection of particulararyl and heteroaryl substituents that extend as conjugated sidechaingroups from the 1,4- or 1,3-phenylene co-monomer can provide forfine-tuning of the spectroscopic properties of the subject deep UVabsorbing polymeric dyes. In some embodiments, the substituted phenyleneco-monomer is selected from the following structures:

wherein

X is O or S;

Z¹ and Z² are independently selected from CR¹² and N; and

each R¹¹ to R¹² is independently hydrogen, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide and a water solubilizing group.

In some cases, the aryl or heteroaryl co-monomer has structure (h):

wherein X is O or S; and R¹¹ is one or more substituents, eachindependently selected from hydrogen, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide and a water solubilizing group.In some cases, the aryl or heteroaryl co-monomer of structure (h) isselected from the following co-monomers:

In certain instance, the aryl or heteroaryl co-monomer is a substitutedor unsubstituted poly-phenylene co-monomer, e.g., of structure (g)described above. In certain instances, the co-monomer of structure (g)is selected from:

wherein R¹¹ is one or more optional substituents each independentlyselected from hydrogen, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy,hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group; and each * is a site forcovalent attachment to the backbone of the polymer.

In certain instances of the aryl or heteroaryl co-monomers describedherein, each R¹¹ to R¹⁴ is independently selected from H, F, methyl,trifluoromethyl, cyano,

wherein:

X is O or S;

m is an integer from 0-10; and

R⁵ is —(CH₂CH₂O)_(p)R′ wherein R′ is H, alkyl or substituted alkyl(e.g., methyl) and p is an integer from 0-40.

In some embodiments, the multichromophore includes a substituted aryl orheteroaryl co-monomer described by one of the following structures:

In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

where n is 1-20 and R′ is H or lower alkyl. In certain instances, n is 3to 12. In some embodiments, the multichromophore includes a substitutedaryl co-monomer described by the following structure:

where each n is independently 1-20 and each R′ is independently H orlower alkyl. In certain embodiments of the substituted aryl orheteroaryl co-monomer structures shown above, n is 3. In some instances,n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Incertain instances, R′ is methyl. In certain instances, R′ is hydrogen.In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

In some embodiments of any of the formula described herein (e.g.,formula (I)-(V)), the multichromophore includes a substituted arylco-monomer (e.g., M1-M3) having on the following structures:

wherein s is 1-50 (e.g., 6-50 6-40, 6-30 or 6-20); and each R¹¹ isindependently H, alkyl or substituted alkyl. In some embodiments, themultichromophore includes a substituted aryl co-monomer including abranched non-ionic WSG described by one of the following structures:

In some embodiments of formulae described herein, e.g., formula(I)-(IV), the aryl or heteroaryl co-monomers, e.g., L¹, L², M¹, M² andM³ are independently selected from one of formulae (XXIII)-(XXVI):

wherein

Cy₂₋₇ is an aryl or heteroaryl group comprising 2 to 7 fused and/orunfused rings;

Y², Y³ and Y⁴ are independently selected from —CR³—, NR³, N, O, S and—C(═O)— and together form a 5 or 6 membered fused aryl or heterarylring;

each R³ is one or more ring substituents independently selected from H,halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano,alkoxy substituted alkoxy and -T¹-Z¹;

R¹ and R² are independently selected from H, halogen, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T¹-Z¹, or R¹ and R² together form a 5- or 6-membered fused aryl,heteroaryl, cycloalkyl or heterocycle ring which can be optionallysubstituted;

Y⁵ is N or CR⁵ and Y⁷ is N or CR⁷;

R⁴-R⁷ are independently selected from H, halogen, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T¹-Z¹;

Z¹ is a chemoselective functional group or a linked signalingchromophore; and

T¹ is a linker.

Cy₂₋₇ is an aryl or heteroaryl group that can include 2, 3, 4, 5, 6 or 7rings which can be fused together to form one fused ring system or maybe unfused (i.e., linked together via single covalent bonds). In somecases, Cy₂₋₇ is includes 2 or 3 unfused aryl and/or heteroaryl rings. Incertain instances, Cy₂₋₇ is composed of 5 and/or 6 membered carbocyclicand/or heterocyclic rings. In certain cases, Cy₂₋₇ is a bicyclic,tricyclic or quadricyclic aryl or heteroaryl group, such as naphthalene,anthracene, acridine, or quinoline, etc.

In certain embodiments of formulae (XXIII)-(XXVI), the co-monomers(e.g., M¹, M² and M³) are independently selected from one of thefollowing structures (a) to (x):

wherein:

each Y⁸ is independently C(R³)₂, —C(R³)₂C(R³)₂—, —C(R³)₂Si(R³)₂—, NR³ orSi(R³)₂;

X is S or O; each R³ is independently H, a water solubilizing group(e.g., as described herein), amino, substituted amino, halogen, cyano,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano,alkoxy, substituted alkoxy and -T¹-Z¹;

R¹ and R² are independently selected from H, halogen, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T¹-Z¹, or R¹ and R² together form a 5- or 6-membered fused aryl,heteroaryl, cycloalkyl or heterocycle ring which can be optionallysubstituted (e.g., with an R³ group);

Z¹ is a chemoselective functional group or a linked signalingchromophore; and

T¹ is a linker.

In certain instances, Y⁸ is C(R³)₂. In certain instances, Y⁸ is—C(R³)₂C(R³)₂—. In certain instances, Y⁸ is NR³. In certain instances,Y⁸ is Si(R³)₂. In certain embodiments of formula (a)-(x), when theco-monomer is a linking co-monomer, at least one of R¹-R⁷ is -T¹-Z¹. Incertain embodiments of formula (a)-(x), the co-monomer includes awater-solubilizing group (e.g., as described herein). In certainembodiments of formula (s), R³ is H, halogen (e.g., F) or alkoxy (e.g.,methoxy). It is understood that in any of the structures of (a)-(x)described herein, a N heteroatom can be included to convert a phenyl orfused benzo ring into a pyridyl or fused pyrido ring. Such heteroatomsubstituted versions of formula (a)-(x) are meant to be included in thepresent disclosure. In some instances of formula (XXIII)-(XXVI), one ormore of the co-monomers have the structure of one of formula (s), wherea benzo ring is replaced with a pyrido ring:

where Y⁸ is independently C(R⁴)₂, —C(R⁴)₂C(R⁴)₂—, NR⁴ or Si(R⁴)₂.

In some instances of formula (XXIII)-(XXVI) and (a)-(x), the co-monomers(M², M³ and M⁴) are independently selected from one of the followingstructures (ba) to (cd):

wherein:

Y⁹ is C(R⁴)₂, —C(R⁴)₂C(R⁴)₂— or Si(R⁴)₂;

X is S or O;

each R⁴ is independently H, a water solubilizing group (e.g., asdescribed herein), alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonicacid, cyano, alkoxy, substituted alkoxy and -T¹-Z¹; and

R¹ and R² are independently selected from H, halogen, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T¹-Z¹, or R¹ and R² together form a 5- or 6-membered fused aryl orheteroaryl ring which can be optionally substituted. In certainembodiments of formula (ba)-(cd), when the co-monomer is a linkingco-monomer, at least one of R⁴ is -T¹-Z¹. In certain embodiments offormula (ba)-(cd), R⁴ includes a water-solubilizing group (e.g., asdescribed herein).

Fused Tricyclic Co-Monomers

Aryl and heteroaryl co-monomers of interest include fused tricyclicco-monomers. A fused tricyclic co-monomer is a co-monomer including atricyclic aromatic group having three fused rings in a configurationwhere two aryl or heteroaryl 6-membered rings are fused to a central 5or 6-membered carbocyclic or heterocyclic ring. In some cases, the fusedtricyclic co-monomer includes two benzo or pyrido rings fused to acentral 5 or 6 membered carbocyclic or heterocyclic ring. The fusedtricyclic co-monomer can be pi-conjugated to adjacent co-monomers of apolymer backbone via any convenient ring atoms of the fused rings. Thecentral 5- or 6-membered ring may be a carbocycle or a heterocycle,aromatic or partially saturated, and may further include a sidechainsubstituent, e.g., a WSG and/or a linker to a chemoselective tag. Abridged biphenyl co-monomer is a fused tricyclic co-monomer having abiphenyl group where the two phenyl rings are further linked with eachother via a central 6 membered carbocyclic or heterocyclic ring. Incertain instances, the fused tricyclic co-monomer is described by thefollowing structure:

where:

Y is C(R³)₂, —C(R³)₂C(R³)₂—, —C(R³)₂Si(R³)₂—, NR³, Si(R³)₂ or Se;

each Z is independently CH, CR or N;

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG; and

each R is independently H or one or more aryl or heteroaryl substituentsand wherein any two convenient R groups are optionally cyclicallylinked. In some cases, each R refers to one or two ring substituentsindependently selected from halogen, sulfonate, alkoxy, substitutedalkoxy, alkyl and substituted alkyl and wherein any two convenient Rgroups are optionally cyclically linked. In certain instances, at leasttwo of Z in each ring is CH or CR. In certain instances, one and onlyone of Z in each ring is N.

In certain instances, the fused tricyclic co-monomer is described by oneof the following structures:

where:

Y is C(R³)₂, —C(R³)₂C(R³)₂—, —C(R³)₂Si(R³)₂—, NR³, Si(R³)₂ or Se;

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG; and

each R is independently H, R³ or one or more aryl or heteroarylsubstituents and wherein any two convenient R groups are optionallycyclically linked. In some cases, each R refers to one or two ringsubstituents independently selected from halogen, sulfonate, alkoxy,substituted alkoxy, alkyl and substituted alkyl and wherein any twoconvenient R groups are optionally cyclically linked. The symbol “*”denotes a site for covalent attachment to the unsaturated backbone of aconjugated polymer, e.g., a π conjugated segment, a terminal group, alinker and a linked specific binding member. It is understood that forany of the formulae described herein which includes a 2,7-pi-conjugatedfused tricyclic co-monomer, an analogous formula including an analogous3,6-pi-conjugated co-monomer could also be depicted, and is meant to beencompassed by the present disclosure. In certain cases, the fusedtricyclic co-monomer is a fluorene co-monomer where Y is C(R³)₂. In somecases, the fused tricyclic co-monomer is a carbazole co-monomer where Yis NR³. In some cases, the fused tricyclic co-monomer is a siloleco-monomer where Y is Si(R³)₂. In some cases, the fused tricyclicco-monomer is a bridged biphenyl co-monomer where Y is —C(R³)₂C(R³)₂— oris —C(R³)₂Si(R³)₂—. In some cases, the fused tricyclic co-monomer is abridged biphenyl co-monomer where Y is —CHR³CHR³—. In certain instancesof any of the fused tricyclic co-monomers described herein, each R isindependently selected from H, halogen, alkoxy, substituted alkoxy,alkyl and substituted alkyl. In certain cases, each R is independentlyselected from H, fluoro, chloro, methoxy, substituted alkoxy, alkyl andsubstituted alkyl.

In certain embodiments of the fused tricyclic co-monomer, the co-monomerincludes two R substituent groups that are cyclically linked to providea carbocyclic or heterocyclic ring A that is optionally furthersubstituted:

wherein Y is C(R³)₂, —C(R³)₂C(R³)₂—, —C(R³)₂Si(R³)₂—, NR³, Si(R³)₂ orSe; and each R³ is independently selected from H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG. In certain cases, the fusedtricyclic co-monomer has the structure:

wherein each R⁸-R⁹ is independently selected from H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG. In some cases of theco-monomer, Y is C(R³)₂.

In certain instances, the fused tricyclic co-monomer is described by oneof the following structures:

wherein:

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG (e.g., as described herein);and each R is independently H, R³ or one or more aryl or heteroarylsubstituents. In some cases, each R refers to one or two ringsubstituents independently selected from halogen, sulfonate, alkoxy,substituted alkoxy, alkyl and substituted alkyl. In some cases, each Ris fluoro or methoxy. In some cases, at least one R³ is a WSG. In somecases, at least one R³ is -L¹¹-Z¹.

In certain instances, the fused tricyclic co-monomer is described by thestructure:

wherein each WSG is a branched or linear water soluble group, and eachR³ is H, alkyl, substituted alkyl. In certain cases, each WSG is alinear WSG (e.g., as described herein). In certain instances, the fusedtricyclic co-monomer is described by the structure:

wherein T⁵ is an optional linker; each s is independently an integerfrom 6 to 100 (e.g., 6 to 50); and each R and R¹¹ is independentlyhydrogen, an alkyl or a substituted alkyl.

A fused 6-5-6 tricyclic co-monomer is a co-monomer including a tricyclicaromatic group having three fused rings in the configuration 6-5-6, i.e.two benzo ring fused to a central 5 membered ring. The 5-membered ringmay be a carbocycle or a heterocycle and may further include a sidechainsubstituent at the ring atom that is not fused to a benzo ring. Incertain instances, the fused 6-5-6 tricyclic co-monomer is described bythe following structure:

where: Z is —C(R¹)₂—, Si(R¹)₂— or —N(R¹)—; each R is independently H orone or more aryl substituents; and each R¹ is independently selectedfrom branched non-ionic water soluble group (WSG), an alkyl, asubstituted alkyl, an aralkyl, a substituted aralkyl, a PEG moiety and-L¹¹-Z¹, where L¹¹ is a linker and Z¹ is a chemoselective tag (e.g., atag including a chemoselective functional group) or a WSG. As used inany of the formulae described herein, * denotes a site for covalentattachment to the unsaturated backbone of a conjugated polymer or an endgroup. In some embodiments, when Z is —N(R¹)—, the fused 6-5-6 tricyclicco-monomer is a carbazole co-monomer. Any convenient carbazoleco-monomers may be utilized in the subject multichromophores. In someembodiments, when Z is —C(R¹)₂—, the fused 6-5-6 tricyclic co-monomer isa fluorene co-monomer. Any convenient fluorene co-monomers may beutilized in the subject multichromophores. In certain instances of thefused 6-5-6 tricyclic co-monomer, each R¹ is selected from a benzylgroup substituted with one, two or more PEG moieties or an alkyl groupsubstituted with two or more PEG moieties. In some embodiments, Z is—Si(R¹)₂—. It is understood that for any of the fluorene co-monomersdescribed herein, also included in the present disclosure is thecorresponding co-monomer where the C atom of Z is replaced with Si. Afluorene co-monomer is a co-monomer including an aromatic group having a9H-fluorene core structure substituted at the 9 position with anyconvenient sidechain substituent(s). In some cases, the fluoreneco-monomer is a 9,9-disubstituted fluorene. The fluorene co-monomer isconjugated to adjacent polymeric backbone groups via any convenientpositions of the fluorene core structure, such as any two positions ofpositions 1-8 (see numbering scheme below). In some embodiments, thefluorene core structure is linked to adjacent groups of the polymerbackbone via the 2 and 7 positions. In certain embodiments, the fluoreneco-monomer is described by the following structure:

where: each R¹ is independently selected from a branched non-ionic watersoluble group (WSG) an alkyl, a substituted alkyl, an aralkyl, asubstituted aralkyl, a PEG moiety and -L¹¹-Z¹, where L¹¹ is a linker andZ¹ is a chemoselective tag (e.g., a tag including a chemoselectivefunctional group) or a WSG. In certain instances of the fluoreneco-monomer, each R¹ is a branched non-ionic water soluble group (WSG).In certain instances of the fluorene co-monomer, each R¹ is selectedfrom a benzyl group substituted with one, two or more PEG moieties or analkyl group substituted with two or more PEG moieties. The Z¹ functionalgroup may find use in covalently linking the multichromophore to anacceptor chromophore (e.g., as described herein). In certain instances,Z¹ includes an amino group for covalently linking to the acceptorchromophore. In certain instances, Z¹ includes an carboxylic acid group,or derivative thereof, for covalently linking to the acceptorchromophore. In certain embodiments, L¹¹ is a branched linker that linksto two or more Z¹ groups (e.g., WSGs). In certain instances, thefluorene co-monomer is further substituted with a R⁵ and/or R⁶substituent located at one, two or more positions selected frompositions 1, 3, 4, 5, 6 and 8, where R⁵ and R⁶ are independentlyselected from a water solubilizing group (WSG) and an aryl substituent(e.g., as described herein).

In certain instances, the fluorene co-monomer is described by thestructure:

where: each R¹ is as defined above; and R⁵ and R⁶ are independentlyselected from H, a water solubilizing group, or an aryl substituent(e.g., as described herein).

In some instances, the fluorene co-monomer is described by thestructure:

where each R² is a alkyl substituted with a water solubilizing group ora branched linker connected to two or more water solubilizing groups(e.g., a PEG-disubstituted benzyl or a PEG substituted alkyl). Incertain instances of the fluorene co-monomer, each R² is a benzyl groupsubstituted with one, two or three PEG moieties (e.g., —O(CH₂CH₂O)_(n)R′where R′ is H or an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16).In certain instances of the fluorene co-monomer, each R² is a benzylgroup substituted with one —O(CH₂CH₂O)_(n)R′ group (e.g., at the 2, 3 or4 position), where R′ is H or an alkyl and n is 1-20, e.g., 3-16 such asn is 8-16. In certain instances of the fluorene co-monomer, each R² is abenzyl group substituted with two —O(CH₂CH₂O)_(n)R′ groups (e.g., at the2,4-, 3,4- or 3,5-positions), where each R′ is independently H or analkyl and each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances of the fluorene co-monomer, each R² is a benzyl groupsubstituted with three —O(CH₂CH₂O)_(n)R′ groups (e.g., at the 2,4,6-,2,4,5- or 3,4,5-positions), where each R′ is independently H or an alkyland each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances of the fluorene co-monomer, each R² is a lower alkylgroup substituted with a trivalent branching group each substituted withtwo PEG moieties (e.g., a —CO—NR″₂ or —O(CH₂R″)₂ trivalent branchinggroup where each R″ is independently a PEG moiety (e.g.,—O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 1-20, e.g., 3-16such as n is 8-16). In certain instances of the fluorene co-monomer,each R² is a branched non-ionic water soluble group (WSG) e.g., asdescribed herein.

In certain embodiments, the fluorene co-monomer is described by thefollowing structure:

where R³ is an alkyl substituted with a water solubilizing group (e.g.,a PEG substituted alkyl) or a branched non-ionic water soluble group(WSG) (e.g., as described herein), and R⁴ is L²-Z² wherein L² is alinker and Z² is a chemoselective tag (e.g., for conjugation to anacceptor chromophore). In some instances, the fluorene co-monomer isdescribed by the structure:

wherein: R³ is a substituent comprising a water solubilizing group(e.g., as described herein) or a branched non-ionic water soluble group(WSG) (e.g., as described herein); R⁴ is L²-Z² wherein L² is a linkerand Z² is a chemoselective tag (e.g., for conjugation to an acceptorchromophore); and R⁵ and R⁶ are independently selected from H, a watersolubilizing group and an aryl substituent (e.g., an alkyl, asubstituted alkyl, an alkoxy, a substituted alkoxy, a halogen or anitro). In certain instances of the fluorene co-monomer, R³ is a loweralkyl group substituted with a trivalent branching group eachsubstituted with two PEG moieties (e.g., a —CO—NR″₂ or —O(CH₂R″)₂trivalent branching group where each R″ is a PEG moiety (e.g.,—O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 6-20, e.g., 8-16such as n is 12-16). In certain instances of the fluorene co-monomer, R³is a branched non-ionic water soluble group (WSG) e.g., as describedherein.

Any of the fluorene co-monomers described herein may be utilized in thesubject multichromophores, e.g., multichromophores of formulae (I)-(IV).In some cases, the multichromophores include, as part of the polymericbackbone, one of the following structures:

where each R³ is independently a water solubilizing group connected viaan optional linker, or an optionally substituted alkyl, aralkyl or arylgroup; Ar is an optionally substituted aryl or heteroaryl group; n is aninteger from 1 to 100,000; and the terminals are sites for covalentattachment to the unsaturated backbone of a conjugated polymer or an endgroup. In certain embodiments, each R³ is independently a substitutedalkyl group. In certain embodiments, each R³ is independently asubstituted aralkyl group. In some cases, each R³ and each Ar areindependently substituted (via an optional linker) with a watersolubilizing group, an acceptor chromophore, a chemoselective functionalgroup or a specific binding moiety.

Any of the co-monomers described herein (e.g., co-monomers of formula(I)-(V), (a)-(k), (a)-(x) and (ba)-(cd)) can include a watersolubilizing group WSG (e.g., as described herein). In some embodiments,the aryl and heteroaryl co-monomer includes a branched non-ionic WSGthat is selected from one of the following structures:

wherein: T⁵ is an optional linker to the co-monomer; each s isindependently an integer from 6 to 50 or 6-30, such as 6 to 20, 11 to20, 12 to 20, or 12 to 16; and each R¹¹ is independently hydrogen, analkyl or a substituted alkyl. In certain cases, each s is independently6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases,each s is 11. In some cases, each s is 12. In some cases, each s is 13.In some cases, each s is 14. In some cases, each s is 15. In some cases,each s is 16. In some cases, each s is 17. In some cases, each s is 18.In some cases, each s is 19. In some cases, each s is 20. In someembodiments, T⁵ is an alkyl linker, such as a C1-C6 alkyl linker. Insome embodiments, T⁵ is a substituted alkyl linker. In some embodiments,T⁵ is an alkoxy linker (e.g., —O-alkyl-). In some embodiments, T⁵ is asubstituted alkoxy.

In certain instances of the co-monomers, the aryl and heteroarylco-monomer includes a WSG (e.g., R³ and/or R⁴) selected from one of thefollowing structures:

wherein: T⁵ is an optional linker; and each s is an integer from 1 to50. In certain instances, each s is independently 1 to 20, such as 3 to20, 3 to 15, 3 to 12, or 6 to 20 or 6 to 12 or 12-16. In certain cases,each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In somecases, each s is 3. In some cases, each s is 4. In some cases, each s is5. In some cases, each s is 6. In some cases, each s is 7. In somecases, each s is 8. In some cases, each s is 9. In some cases, each s is10. In some cases, each s is 11. In some embodiments, T⁵ is an alkyllinker, such as a C1-C6 alkyl linker. In some embodiments, T⁵ is asubstituted alkyl linker. In some embodiments, T⁵ is an alkoxy linker(e.g., —O-alkyl-). In some embodiments, T⁵ is a substituted alkoxy.

In certain embodiments of the formulae described herein, e.g., formulae(I)-(IV), the co-monomers (e.g., M¹, M² and/or M³) are independentlyselected from one of the following structures:

where each s is independently 1-30, such as 6-30, such as 6 to 20, 11 to20, 12 to 20, or 12 to 16. In certain cases, each s is independently 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases,each s is 4. In some cases, each s is 5. In some cases, each s is 6. Insome cases, each s is 7. In some cases, each s is 8. In some cases, eachs is 9. In some cases, each s is 10. In some cases, each s is 11. Insome cases, each s is 12. In some cases, each s is 13. In some cases,each s is 14. In some cases, each s is 15. In some cases, each s is 16.In some cases, each s is 17. In some cases, each s is 18. In some cases,each s is 19. In some cases, each s is 20.

In some instances, the multichromophore (e.g., of formulae (I)-(IV))includes a co-monomer (e.g., L¹) linked to a signaling chromophorehaving the one of the following structures:

wherein WSG is an optional water soluble group; T² is a linker; and Dyeis the signaling chromophore.

In certain embodiments, the aryl or heteroaryl co-monomer is anoptionally substituted co-monomer selected from 2,1,3-benzothiadiazole,2,1,3-benzoxadiazole, benzoxidazole, benzoselenadiazole,benzotellurodiazole, naphthoselenadiazole,4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines,perylene, perylene diimides, diketopyrrolopyrrole, thienopyrazine lowbandgap commercial dyes, olefins, and cyano-substituted olefins andisomers thereof. In some instances, aryl and heteroaryl co-monomerswhich find use in the subject multichromophores are selected from a′-m′having the structure:

wherein *=a site for covalent attachment to unsaturated backbone andeach R is independently H, a non-ionic side group capable of impartingsolubility in water (e.g., a WSG), or -L²-Z², where L² is a linker andZ² is a chemoselective tag or a linked metal complex. In certaininstances of a′-m′, each R is H or a water solubilizing group (e.g., asdescribed herein). In certain cases, each R is an alkyl or a benzylsubstituted with one or more (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x isindependently an integer from 0-20, each y is independently an integerfrom 0 to 50. In certain instances of a′-m′, each R is(CH₂)₃(OCH₂CH₂)₁₁OCH₃. In certain instances of a′-m′, each R is H or anon-ionic branched WSG, e.g., as described herein.

In some embodiments, the multichromophore includes anabsorbance-modifying co-monomer having the following structure:

where X is O or S, R⁴¹ and R⁴² are each independently, H, halogen, aWSG, an alkyl, a substituted alkyl, an alkoxy and a substituted alkoxy.In certain instances, X is O. In some instances, X is S. In certainembodiments, X is O and R⁴¹ and R⁴² are each H. In certain embodiments,X is S and R⁴¹ and R⁴² are each H.

Any of the absorbance-modifying co-monomers described above may beutilized in the subject multichromophores, e.g., multichromophores offormulae (I)-(IV).

Water Solubilizing Groups

The repeat units of the subject polymeric dyes can be substituted at anyconvenient positions with a water solubilizing group. As used herein,the terms “water solubilizing group”, “water soluble group” and WSG areused interchangeably and refer to a group or substituent that is wellsolvated in aqueous environments e.g., under physiological conditions,and that imparts improved water solubility upon the molecules to whichit is attached. In some embodiments, a WSG increases the solubility ofthe multichromophore in a predominantly aqueous solution, as compared toa control multichromophore which lacks the WSG. The water solubilizinggroups may be any convenient hydrophilic group that is well solvated inaqueous environments.

A water soluble polymeric dye of the present disclosure has solubilityunder aqueous conditions that makes it especially suitable forapplication to a variety of biological assays. The subject water solublepolymers, and conjugates thereof, can be resistant to undesirableaggregation which provides advantageous fluorescence and spectroscopicproperties in various biological assays. Aggregation of dyes isundesirable because it can lead to reduced fluorescent signals, e.g.,via aggregation-caused quenching of dye fluorescence. The subjectwater-soluble conjugated polymer dyes can be used as fluorescentreporters for a variety of biosensors and provide signals of exceptionalbrightness with a range of options for excitation and emissionwavelength for applications such as Flow Cytometry, and imaging.

In certain instances, a water soluble polymeric dye has a solubility inwater or buffer of 10 mg/mL or more, such as 20 mg/mL or more, 30 mg/mLor more, 40 mg/mL or more, 50 mg/mL or more, 60 mg/mL or more, 70 mg/mLor more, 80 mg/mL or more, 90 mg/mL or more, 100 mg/mL or more, or evenmore. It is understood that water soluble polymeric dyes may beconfigured to, under certain conditions, form discrete water solvatednanoparticles in aqueous systems. In certain cases, the water solvatednanoparticles are resistant to aggregation and find use in a variety ofbiological assays.

Any convenient water solubilizing groups (WSG's) may be included in themultichromophores described herein (e.g., multichromophores of formulae(I)-(IV)) to provide for increased water-solubility. While the increasein solubility may vary, in some instances the increase (as compared tothe compound without the WSG(s)) is 2 fold or more, e.g., 5 fold, 10fold, 25 fold, 50 fold, 100 fold or more. In some cases, the hydrophilicwater solubilizing group is charged, e.g., positively or negativelycharged. In certain cases, the hydrophilic water solubilizing group is aneutral hydrophilic group. In some embodiments, the WSG is branched(e.g., as described herein). In certain instances, the WSG is linear. Insome embodiments, the WSG is a hydrophilic polymer, e.g., a polyethyleneglycol, a modified PEG, a peptide sequence, a peptoid, a carbohydrate,an oxazoline, a polyol, a dendron, a dendritic polyglycerol, acellulose, a chitosan, or a derivative thereof. Water solubilizinggroups of interest include, but are not limited to, carboxylate,phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester,polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, aminoacid, ammonium, guanidinium, pyridinium, polyamine and sulfonium,polyalcohols, straight chain or cyclic saccharides, primary, secondary,tertiary, or quaternary amines and polyamines, phosphonate groups,phosphinate groups, ascorbate groups, glycols, including, polyethers,—COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺, Y′, (CH₂CH₂O)_(p)R and mixtures thereof,where Y′ can be any halogen, sulfate, sulfonate, or oxygen containinganion, p can be 1 to 500, each R can be independently H or an alkyl(such as methyl) and M′ can be a cationic counterion or hydrogen,—(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy), —(CH₂CH₂O)_(yy)CH₂CH₂X—,—X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, and polyethylene glycol, wherein yy isselected from 1 to 1000, X is selected from O, S, and NR^(ZZ), andR^(ZZ) and R^(YY) are independently selected from H and C₁₋₃ alkyl. Insome cases, a WSG is (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x isindependently an integer from 0-20, each y is independently an integerfrom 0 to 50. In some cases, the water solubilizing group includes anon-ionic polymer (e.g., a PEG polymer) substituted at the terminal withan ionic group (e.g., a sulfonate).

A WSG can include a water-soluble polymer. Water-soluble polymers ofinterest include, but are not limited to, polyalkylene oxide basedpolymers, such as polyethylene glycol “PEG” (See. e.g., “Poly(ethyleneglycol) Chemistry: Biotechnical and Biomedical Applications”, J. M.Harris, Ed., Plenum Press, New York, N.Y. (1992); and “Poly(ethyleneglycol) Chemistry and Biological Applications”, J. M. Harris and S.Zalipsky, Eds., ACS (1997); and International Patent Applications: WO90/13540, WO 92/00748, WO 92/16555, WO 94/04193, WO 94/14758, WO94/17039, WO 94/18247, WO 94/28937, WO 95/11924, WO 96/00080, WO96/23794, WO 98/07713, WO 98/41562, WO 98/48837, WO 99/30727, WO99/32134, WO 99/33483, WO 99/53951, WO 01/26692, WO 95/13312, WO96/21469, WO 97/03106, WO 99/45964, and U.S. Pat. Nos. 4,179,337;5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264;5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808;5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027;5,446,090; 5,470,829;

5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528;5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110;5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784;5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442;5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237;6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087;6,180,095; 6,194,580; 6,214,966).

Examples of water soluble polymers of interest include, but are notlimited to, those containing a polyalkylene oxide, polyamide alkyleneoxide, or derivatives thereof, including polyalkylene oxide andpolyamide alkylene oxide comprising an ethylene oxide repeat unit of theformula —(CH₂—CH₂—O)—. Further examples of polymers of interest includea polyamide having a molecular weight greater than 1,000 Daltons of theformula —[C(O)—X—C(O)—NH—Y—NH]n- or —[NH—Y—NH—C(O)—X—C(O)]_(n)—, where Xand Y are divalent radicals that may be the same or different and may bebranched or linear, and n is a discrete integer from 2-100, such as from2 to 50, and where either or both of X and Y comprises a biocompatible,substantially non-antigenic water-soluble repeat unit that may be linearor branched. Further examples of water-soluble repeat units comprise anethylene oxide of the formula —(CH₂—CH₂—O)— or —(O—CH₂—CH₂)—. The numberof such water-soluble repeat units can vary significantly, with thenumber of such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200,2 to 100, 6-100, for example from 2 to 50 or 6 to 50, such as 6 to 40, 6to 30, 6 to 20, 8 to 20 or 10 to 20, e.g., 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. An example of an embodiment is one in which one orboth of X and Y is selected from: —((CH₂)_(n1)—(CH₂—CH₂—O)_(n2)—(CH₂)—or —((CH₂)_(n1)—(O—CH₂—CH₂)_(n2)—(CH₂)_(n1)—), where n1 is 1 to 6, 1 to5, 1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to10, 2 to 8, or 2 to 5. A further example of an embodiment is one inwhich X is —(CH₂—CH₂)—, and where Y is —(CH₂—(CH₂—CH₂—O)₃—CH₂—CH₂—CH₂)—or —(CH₂—CH₂—CH₂—(O—CH₂—CH₂)₃—CH₂)—. In certain instances, theco-monomer includes 2, 3, or 4 WSG each independently having the formula—(CH₂)_(n1)—(CH₂—CH₂—O)_(n2)R′ where R′ is H, C1-6alkyl or substitutedC1-6alkyl, n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and n2 is 2 to 50,such as 6 to 40, 6 to 30, 6 to 20, 8 to 20 or 10 to 20, e.g., 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20.

As used herein a modified polymer, such as a modified PEG, refers towater soluble polymers that have been modified or derivatized at eitheror both terminals, e.g., to include a terminal substituent (e.g., aterminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc)and/or a terminal linking functional group (e.g., an amino gorcarboxylic acid group suitable for attachment via amide bond formation)suitable for attached of the polymer to the multichromophore (e.g., viaa branching group). The subject water soluble polymers can be adapted toinclude any convenient linking groups.

It is understood that in some cases, the water soluble polymer caninclude some dispersity with respect to polymer length, depending on themethod of preparation and/or purification of the polymeric startingmaterials. In some instances, the subject water soluble polymers aremonodisperse. In some instances, the subject water soluble polymers aresubstantially monodisperse, e.g., include 20 wt % or less of non-targetspecies, such as 15 wt % or less, 10 wt % or less, 5 wt % or less, 2 wt% or less or 1 wt % or less.

The water soluble polymer can include one or more spacers or linkers.Examples of spacers or linkers include linear or branched moietiescomprising one or more repeat units employed in a water-soluble polymer,diamino and or diacid units, natural or unnatural amino acids orderivatives thereof, as well as aliphatic moieties, including alkyl,aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which can contain,for example, up to 18 carbon atoms or even an additional polymer chain.

The water soluble polymer moiety, or one or more of the spacers orlinkers of the polymer moiety when present, may include polymer chainsor units that are biostable or biodegradable. For example, polymers withrepeat linkages have varying degrees of stability under physiologicalconditions depending on bond lability. Polymers with such bonds can becategorized by their relative rates of hydrolysis under physiologicalconditions based on known hydrolysis rates of low molecular weightanalogs, e.g., from less stable to more stable, e.g., polyurethanes(—NH—C(O)—O—)>polyorthoesters (—O—C((OR)(R′))—O—)>polyamides(—C(O)—NH—). Similarly, the linkage systems attaching a water-solublepolymer to a target molecule may be biostable or biodegradable, e.g.,from less stable to more stable: carbonate (—O—C(O)—O—)>ester(—C(O)—O—)>urethane (—NH—C(O)—O—)>orthoester (—O—C((OR)(R′))—O—)>amide(—C(O)—NH—). In general, it may be desirable to avoid use of a sulfatedpolysaccharide, depending on the lability of the sulfate group. Inaddition, it may be less desirable to use polycarbonates and polyesters.These bonds are provided by way of example, and are not intended tolimit the types of bonds employable in the polymer chains or linkagesystems of the water-soluble polymers useful in the modified aldehydetagged polypeptides disclosed herein.

Branched Non-Ionic Water Soluble Groups

In some cases, a WSG is a branched non-ionic water soluble group. Abranched non-ionic water soluble group can be attached to a co-monomerat any convenient location to provide a sidechain group that projectsaway from the backbone of the conjugated polymer to provide highsolvation in an aqueous environment. In some cases, the branchednon-ionic water soluble groups (WSG) comprise two or more water solublepolymers (e.g., as described herein) each having 6-100, such as 6-50,6-40 or 6-30 monomeric units.

A branched non-ionic water soluble group (WSG) comprises a branchinggroup that is linked to the co-monomer to which it is attached andprovides further linkages to two, three or more non-ionic water solublepolymers. In some instances, the two or more water soluble polymers thatare utilized in the WSG are polyethylene glycol (PEG) groups or modifiedPEG groups. Any convenient branching groups can be utilized that providefor 3 or more points of attachment (e.g., 4 or more), such as one pointof attachment to the co-monomer of the conjugated polymer backbone andtwo or more attachments (e.g., 2, 3, 4 or more) to non-ionic watersoluble polymers. The branching group can be attached to the co-monomerand the non-ionic water soluble polymers via optional linkers utilizingany convenient linking functional groups and linking chemistries. Incertain instances, the branching group is non-aromatic. In certaininstances, the branching group is cyclic. In some cases, the branchinggroup is a substituted aryl or heteroaryl ring, e.g., a trisubstitutedor tetrasubstituted aryl ring, such as a trisubstituted ortetrasubstituted phenyl ring, or a trisubstituted or tetrasubstitutedheteroaryl ring, such as a trisubstituted or tetrasubstituted pyridylring. In certain instances, the branching group is acyclic. In someinstances, the branching group is an atom, e.g., C, Si, N. In certaininstances, the branching group is a linking functional group such as anamido or a sulfonamide group. In certain instances, the branching groupis an amino acid residue or a branched linker, such as a glycerol or anamino-glycerol.

In some instances, the branched non-ionic WSG is independently selectedfrom: —Y¹—(CH₂CH₂O)_(r)—R′; —Y¹—O—CH[(CH₂)_(q)—O—(CH₂CH₂O)_(r)—R′]₂; and—Y¹—CH₂-Ph(Y¹—(CH₂CH₂O)_(r)—R′)_(s); wherein Y¹ is selected from acovalent bond, —O—, —CONH—, —NHCO—, NHSO₂—, —SO₂—NH—, —CONR—, —NRCO—,NRSO₂—, —SO₂—NR— (e.g., where R is alkyl or substituted alkyl),—(CH₂)_(q)—SO₂—NH—, —(CH₂)_(q)—CONH— and —(CH₂)_(q)—O—, q and r are eachindependently an integer from 1 to 50, s is 1, 2 or 3 and each R′ isindependently H, alkyl (e.g., methyl) or substituted alkyl.

In some instances, the branched non-ionic WSG has one of the followingformulae:

wherein:

each B¹ and B² are independently a branching group;

each W¹ is independently a non-ionic water soluble polymer, e.g.,comprising 6 or more monomeric units;

T³ is an optional linker to the fused 6-5-6 tricyclic co-monomer; and

each p and q are independently 0 or 1, wherein if present, each T¹ andeach T² are independently a linker. In certain instances, each W¹ isindependently a PEG or modified PEG polymer. In certain instances, eachW¹ is independently selected from a substituted alkyl, a PEG or modifiedPEG group and a WSG. In certain instances, each W¹ is independently aPEG or modified PEG polymer of 6-30 monomeric units, such as 6-24 or10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units.

In some instances, the branched non-ionic WSG has the following formula:

wherein:

each B¹ is a branching group;

each W¹ is independently a non-ionic water soluble polymer, e.g.,comprising 6 or more monomeric units;

T³ is an optional linker to the fused 6-5-6 tricyclic co-monomer; and

each p is independently 0 or 1, wherein if present, each T¹ isindependently a linker. In certain instances, each W¹ is independently aPEG or modified PEG polymer. In certain instances, each W¹ isindependently selected from a substituted alkyl, a PEG or modified PEGgroup and a WSG. In certain instances, each W¹ is independently a PEG ormodified PEG polymer of 6-30 monomeric units, such as 6-24 or 10-30,10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units. In someembodiments of the branched non-ionic WSG, B¹ is a tetra-substitutedaryl group (e.g., a 1,3,4,5-phenyl).

In some embodiments of the branched non-ionic WSG (e.g., as depicted inthe formulae above), B¹ is selected from CH, N, C(═O)N, SO₂N, atri-substituted aryl group (e.g., a 1,3,5-phenyl), a tetra-substitutedaryl group, and a tri-substituted heteroaryl group. In some embodimentsof the branched non-ionic WSG, each p is 0. In some embodiments of thebranched non-ionic WSG, p is 1, and each T¹ is selected from—(CH₂)_(n)—O—, —O—(CH₂)_(n)—, —(CH₂)_(n)— and —O—, wherein n is from 1to 12, e.g., 1 to 6. In some embodiments of the branched non-ionic WSG,each T² and/or T³ is independently a C1-C12-alkyl linker, e.g., aC1-C6-alkyl linker, wherein one or more backbone atoms are optionallysubstituted with a heteroatom.

In some embodiments, the branched non-ionic WSG is selected from one ofthe following structures:

wherein:

T⁵ is an optional linker to the co-monomer (e.g., the fused 6-5-6tricyclic co-monomer);

T⁶ is a linker;

each s is independently an integer from 6 to 100 (e.g., 6 to 50); and

each R¹¹ is independently hydrogen, an alkyl or a substituted alkyl.

In certain instances, each s is independently 6 to 30, such as 6 to 24,6 to 20, 11 to 20, 12 to 20, 12 to 18 or 12 to 16. In certain instances,each s is independently 6 to 30, such as 6 to 24, 8 to 24, 10 to 24, 12to 24, 13 to 24, 14 to 24, 15 to 22 or 16 to 20. In some cases, each sis independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or24. In some embodiments, each s is independently 7 or more, such as 8, 9or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15or more, or even more, and in some cases, have up to 50 monomeric units,such as up to 40, up to 30 or up to 24 monomeric units. In someinstances, each s is independently 6-30 monomeric units, such as 6-24 or10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units. Insome cases, each s is the same. In some embodiments of the branchednon-ionic WSG, T⁵ and/or T⁶ is a C1-C12-alkyl linker, e.g., aC1-C6-alkyl linker, wherein one or more backbone atoms are optionallysubstituted with a heteroatom (e.g., an —O—). In some embodiments of thebranched non-ionic WSG, each R¹¹ is H. In some embodiments of thebranched non-ionic WSG, each R¹¹ is methyl.

In some embodiments, the π-conjugated segment includes a fluoreneco-monomer substituted with two branched non-ionic WSG groups (e.g., asdescribed herein) (e.g., disubstituted at the 9 position). In someembodiments of formula (I), F¹ is a fluorene co-monomer substituted withtwo linear non-ionic WSG groups (e.g., as described herein) (e.g.,disubstituted at the 9 position). In some embodiments, the fluoreneco-monomer is of the structure:

wherein s is 6-60; t is 0-10 (e.g., 0-6 such as 0, 1, 2, or 3); and R¹¹is H, alkyl or substituted alkyl. In certain cases, each R¹¹ is methyl.

In some embodiments of the formulae, the co-monomer includes asubstituent selected from (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x isindependently an integer from 0-20, each y is independently an integerfrom 0 to 50; and a benzyl optionally substituted with one or morehalogen, hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(z)OCH₃ where each z isindependently an integer from 0 to 50. In some instances, thesubstituent is (CH₂)₃(OCH₂CH₂)_(y)OCH₃. In some embodiments, one or moreof the substituents is a benzyl substituted with at least one WSG groups(e.g., one or two WSG groups) selected from (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃where each x is independently an integer from 0-20 and each y isindependently an integer from 0 to 50.

Multiple WSGs may be included at a single location in the subjectmultichromophores via a branching linker. In certain embodiments, thebranching linker is an aralkyl substituent, further di-substituted withwater solubilizing groups. As such, in some cases, the branching linkergroup is a substituent of the multichromophore that connects themultichromophore to two or more water solubilizing groups. In certainembodiments, the branching linker is an amino acid, e.g., a lysine aminoacid that is connected to three groups via the amino and carboxylic acidgroups. In some cases, the incorporation of multiple WSGs via branchinglinkers imparts a desirable solubility on the multichromophore. In someinstances, the WSG is a non-ionic sidechain group capable of impartingsolubility in water in excess of 50 mg/mL. In some instances, the WSG isa non-ionic sidechain group capable of imparting solubility in water inexcess of 100 mg/mL. In some embodiments, the multichromophore includessubstituent(s) selected from the group consisting of, an alkyl, anaralkyl and a heterocyclic group, each group further substituted with ainclude water solubilizing groups hydrophilic polymer group, such as apolyethylglycol (PEG) (e.g., a PEG group of 2-20 units).

In certain instances of any one of the formulae described herein, one ormore of the co-monomers is substituted with a WSG and/or a linkedsignaling chromophore. Any convenient WSG can adapted for inclusion intoa co-monomer of the subject polymeric dyes. In certain instances of anyone of the formulae described herein, one or more of the co-monomers issubstituted with a WSG that is independently selected from one of thefollowing structures:

wherein: each R¹¹ is H, an alkyl (e.g., methyl) or a substituted alkyl;T⁵ is an optional linker; T⁶ is an linker; and each s is independentlyan integer from 1 to 100 (e.g., 1 to 50, or 6 to 50). In certaininstances, each s is independently 1 to 50, such as 3 to 40, 3 to 30, 3to 20, 3 to 15, 3 to 12, or 6 to 12. In certain cases, each s isindependently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20. In some cases, each s is 3. In some cases, each s is 4. Insome cases, each s is 5. In some cases, each s is 6. In some cases, eachs is 7. In some cases, each s is 8. In some cases, each s is 9. In somecases, each s is 10. In some cases, each s is 11. In some cases, each sis 12. In some cases, each s is 14. In some cases, each s is 16. In someembodiments, T⁶ is an alkyl linker, such as a C1-C12 or C1-C6 alkyllinker. In some embodiments, T⁶ is a substituted alkyl linker. In someembodiments, T⁶ is an alkoxy linker (e.g., —O-alkyl-). In someembodiments, T⁶ is a substituted alkoxy. It is understood thathydroxy-terminated PEG chains instead of methoxy-terminated PEG chainsmay be utilized in any of the WSG groups described herein. In someembodiments, each R¹¹ is H. In some embodiments, each R¹¹ is methyl.

In certain instances of any one of the formulae described herein, one ormore of the co-monomers is substituted with a WSG that is independentlyselected from one of the following structures:

wherein: T⁵ is an optional linker; and each s is an integer from 1 to50. In certain instances of the WSG, T⁵ is absent. In certain instances,each s of the WSG is independently 1 to 20, such as 3 to 20, 3 to 15, 3to 12, or 6 to 12. In certain cases, each s of the WSG is independently1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some cases, each s of theWSG is 3. In some cases, each s of the WSG is 4. In some cases, each sof the WSG is 5. In some cases, each s is 6. In some cases, each s ofthe WSG is 7. In some cases, each s of the WSG is 8. In some cases, eachs of the WSG is 9. In some cases, each s of the WSG is 10. In somecases, each s of the WSG is 11. In some cases, each s of the WSG is 12.In some cases, each s of the WSG is 14. In some cases, each s of the WSGis 16. In some embodiments, T⁵ is an alkyl linker, such as a C1-C12 orC1-C6 alkyl linker. In some embodiments, T⁵ is a substituted alkyllinker. In some embodiments, T⁵ is an alkoxy linker (e.g., —O-alkyl-).In some embodiments, T⁵ is a substituted alkoxy. It is understood thathydroxy-terminated PEG chains instead of methoxy-terminated PEG chainsmay be utilized in any of the WSG groups described above. In certaininstances of any one of the formulae described herein, one or more ofthe co-monomers is substituted with a WSG that is a dendron selectedfrom one of the following structures:

In certain instances of any one of the formulae described herein, one ormore of the co-monomers is substituted with a WSG that is a polyolselected from one of the following structures:

In certain instances of any one of the formulae described herein, one ormore of the co-monomers is substituted with WSG that is an oxazoline ofthe following structure:

In certain instances of any one of the formulae described herein, one ormore of the co-monomers is substituted with a WSG that is a peptoidselected from one of the following structures:

Polymeric Dyes

Further details of the subject polymeric dyes are provided in theexemplary formula below. In some embodiments, the subject polymeric dyehas the structure of formula (I):

wherein:

each M¹ is independently selected from a substituted or unsubstitutedaryl co-monomer, a substituted or unsubstituted heteroaryl co-monomer,an alkynyl co-monomer and a substituted or unsubstituted alkenylco-monomer (e.g., as described herein);

each M² is independently a conjugation-modifying repeat unit (e.g., asdescribed herein);

L¹ and L² are each independently a linker unit;

Z¹ is a linked chemoselective tag or a linked signaling chromophore(—Z¹);

Z² is a branched π-conjugated segment;

a-h are each independently 0 or 1, wherein a+b≥1 and b+e+h≥1;

n is from 1 to 10,000;

m and o are each independently from 0 to 10,000;

p is from 1 to 100,000; and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member. Incertain instances of formula (I), m is 0. In certain instances offormula (I), m is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10.In certain instances of formula (I), o is 0. In certain instances offormula (I), o is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10.In certain instances of formula (I), d is 1. In certain instances offormula (I), g is 1. In certain instances of formula (I), a+b=2.

In some embodiments of formula (I), M² is a conjugation-modifying repeatunit of formula (XI) (e.g., as described herein). As such, in someembodiments of formula (I), the subject polymeric dye has the structureof formula (II):

wherein:

M¹ and M³ are independently selected from an aryl co-monomer, aheteroaryl co-monomer, an alkynyl co-monomer and an alkenyl co-monomer,each substituted or unsubstituted;

Z is selected from a covalent bond, a saturated atom or group and afunctional group;

R¹¹ and R¹² are each independently selected from H, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group;

a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+e≥1.

In certain instances of formula (II), m is 0. In certain instances offormula (II), m is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10.In certain instances of formula (II), a is 0. In certain instances offormula (II), a is 1. In certain instances of formula (II), b is 1. Incertain instances of formula (II), c is 0. In certain instances offormula (II), e is 1. In certain instances of formula (II), c is 1. Incertain instances of formula (II), a+b=2. In certain instances offormula (II), b+e=2. In certain instances of formula (II), a+c=0.

In some embodiments of formula (I), the subject polymeric dye has thestructure of formula (III):

wherein:

L¹ is a linker unit comprising an aryl co-monomer, a heteroarylco-monomer, an alkynyl co-monomer and an alkenyl co-monomer (e.g., asdescribed herein);

Z¹ is a linked chemoselective tag or a linked signaling chromophore;

Z is selected from a covalent bond, a saturated atom or group and afunctional group (e.g., as described herein);

R¹¹ and R¹² are each independently selected from H, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group;

a, b, d and e are each independently 0 or 1, wherein a+b≥1 and d+e≥1. Incertain instances of formula (III), m is 0. In certain instances offormula (III), n is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to10. In certain instances of formula (III), m is 1 to 10,000, such as 1to 1000, 1 to 100, or 1 to 10. In certain instances of formula (III), ais 1. In certain instances of formula (III), b is 1. In certaininstances of formula (III), d is 1. In certain instances of formula(III), e is 1. In certain instances of formula (III), a+b=2.

In certain instances of formula (III), Z¹ is a linked chemoselectivetag. In certain instances of formula (III), Z¹ is a linked signalingchromophore, and the polymeric dye can be referred to as a polymerictandem dye.

Also provided are polymeric dyes that include a branching point in theconjugated polymer backbone, to which a pi-conjugated segment ofco-monomers can be linked. This branched pi-conjugated segment ofco-monomers can be pi-conjugated to the main backbone of the polymer, orcan be separated from the backbone, e.g., via a conjugation-modifyingrepeat unit. In some embodiments of formula (I), the subject polymericdye has the structure of formula (I), wherein a, b, g and h are each 1;and Z² has is a branched π-conjugated segment of formula (IV):

wherein M¹, M², L¹, Z¹ are as defined for formula (I); and G³ is aterminal group, a π-conjugated segment, a linker or a linked specificbinding member. In certain instances, the polymeric dye is of formula(I) wherein a, b, d, e, g, h are each 1; and Z¹ is a linked functionalsignaling chromophore.

In some embodiments of formula (I), the subject polymeric dye has thestructure of formula (V):

wherein

Z is selected from a covalent bond, a saturated atom or group and afunctional group (e.g., as described herein);

R¹-R³, R¹¹ and R¹² are each independently selected from H, alkyl,substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen,sulfonate, sulfonamide, substituted sulfonamide, carboxy, carboxyamide,substituted carboxyamide, a water solubilizing group, a linkedchemoselective tag and a linked signalling chromophore;

a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

n is from 1 to 10,000;

m is from 0 to 10,000;

p is from 1 to 10,000; and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member. Incertain instances of formula (V), R¹-R³ are each independently a watersolubilizing group; and R² and R³ are the same.

In certain instances of formula (V), R¹ and R² are each independently awater solubilizing group; and R³ is a linked chemoselective tag or alinked signalling chromophore. In certain instances of formula (V), m is0. In certain instances of formula (V), n is 1 to 10,000, such as 1 to1000, 1 to 100, or 1 to 10. In certain instances of formula (V), n is 2to 10,000, such as 2 to 1000, 2 to 100, or 2 to 10. In certain instancesof formula (V), m is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to10. In certain instances of formula (V), m is 2 to 10,000, such as 2 to1000, 2 to 100, or 2 to 10. In certain instances of formula (V), a is 1.In certain instances of formula (V), b is 1. In certain instances offormula (V), c is 1. In certain instances of formula (V), e is 1. Incertain instances of formula (V), a+b=2. Any convenient embodiments ofthe Z groups of a conjugation-modifying repeat unit described herein canbe utilized in a polymeric dye of formula (V). Any convenientembodiments of the WSG described herein can be utilized as R¹-R³, R¹¹and R¹² groups in a polymeric dye of formula (V).

In certain instances of formula (V), the subject polymeric dye has theformula (VI):

wherein:

Dye is the linked signaling chromophore and L is a linker; WSG1 and WSG2are each independently a water solubilizing group (WSG) (e.g., asdescribed herein);

Z is selected from —S—, —S(O)—, —SO₂—, —SO₂NR′—, —S(O)O—, —O—, —C(O)—,—C(O)NR′—, —C(O)O—, —OC(O)NR′—, —OC(O)O—, —NR′C(O)NR′—, —C(R⁵)(R⁶)—,—Si(R⁵)(R⁶)—, —N(R′)—, an alkyl linker (e.g., C₂-C₁₂, such as C₂-C₆substituted or unsubstituted alkyl linker) and a polyethylene glycollinker;

each R′, R⁵ and R⁶ is independently selected from H, alkyl, substitutedalkyl, and wherein R⁵ and/or R⁶ is optionally cyclically linked to R¹²or R¹² to provide a benzo-f used carbocycle or heterocycle ring; and

x and y represent mol % values. In some cases, y is 1-25 mol % and x is99 mol % or less.

In certain instances of formula (VI), Z is selected from —S—, —S(O)—,—SO₂—, —O—, —C(O)—, —C(O)NR′—, —OC(O)NR′—, —OC(O)O— and —NR′C(O)NR′—. Incertain cases of formula (VI), Z is —SO₂—. In some instances of formula(VI), Z is C(R⁵)(R⁶)— or —Si(R⁵)(R⁶)—. In some instances of formula(VI), each WSG¹ and WSG² are each independently a water solubilizinggroup selected from:

wherein m is 0-10 (such as 1-6, e.g., 1, 2, 3, 4, 5 or 6) and each R⁵ isindependently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50(such as 6-50, 6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20) and R′ is H, an alkyl or a substitutedalkyl; and X is O. FIGS. 3 and 6C show exemplary polymeric dyes offormula (V) and (VI).

In certain instances of formula (V), the subject polymeric dye has theformula (VII):

wherein:

R¹-R⁴ are each independently selected from H, alkyl, substituted alkyl,alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide, a water solubilizing group (WSG) (e.g., as describedherein), a linked chemoselective tag and a linked signallingchromophore;

a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

x and y represent mol % values;

p is from 1 to 10,000; and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member. Incertain instances of formula (VII), R¹-R³ are each independently a watersolubilizing group; and R² and R³ are the same. In certain instances offormula (VII), R³ or R⁴ is a linked chemoselective tag or linkedsignalling chromophore. In some cases, y is 1-25 mol % and x is 99 mol %or less.

In certain instances of formula (V), the subject polymeric dye has theformula (VIII):

wherein:

Dye is the linked signaling chromophore and L is a linker; each WSG1,each WSG2 and WSG3 are each independently a water solubilizing group(WSG) (e.g., as described herein); and x and y represent mol % values.In some cases, y is 1-25 mol % and x is 99 mol % or less.

In certain instances of formula (VIII), WSG1, each WSG2 and WSG3 areindependently a water solubilizing group selected from:

wherein m is 0-10 (such as 1-6, e.g., 1, 2, 3, 4, 5 or 6) and each R⁵ isindependently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50(such as 6-50, 6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20) and R′ is H, an alkyl or a substitutedalkyl; and X is O. FIGS. 4 and 6A-B show exemplary polymeric dyes offormula (V), (VII) and (VIII).Polymeric Tandem Dyes

The water soluble light harvesting multichromophore can itself befluorescent and capable of transferring energy to a linked acceptorsignaling chromophore. As such, the subject polymeric tandem dyesfurther include a covalently linked acceptor signaling chromophore inenergy-receiving proximity to the donor water solvated light harvestingmultichromophore. As such, excitation of the multichromophore donorleads to energy transfer to and emission from the covalently attachedacceptor signaling chromophore. The number of signaling chromophoreacceptor units that are linked to the donor water solvated lightharvesting multichromophore may vary, where in some instances the numberranges from 1 mol % to 50 mol %, such as from 5 mol % to 25 mol % orfrom 10 mol % to 25 mol %.

Mechanisms for energy transfer from the fluorescent water solvated lightharvesting multichromophore donor to the linked acceptor signalingchromophroe include, for example, resonant energy transfer (e.g.,Förster (or fluorescence) resonance energy transfer, FRET), quantumcharge exchange (Dexter energy transfer) and the like. In someinstances, these energy transfer mechanisms are relatively short range;that is, close proximity of the light harvesting multichromophore systemto the acceptor provides for efficient energy transfer. In someinstances, under conditions for efficient energy transfer, amplificationof the emission from the acceptor occurs where the emission from theluminescent signaling chromophore is more intense when the incidentlight (the “pump light”) is at a wavelength which is absorbed by thelight harvesting multichromophore than when the luminescent signalingchromophore is directly excited by the pump light.

By “efficient” energy transfer is meant 10% or more, such as 20% or moreor 30% or more, of the energy harvested by the donor is transferred tothe acceptor. By “amplification” is meant that the signal from thesignaling chromophore is 1.5× or greater when excited by energy transferfrom the donor light harvesting multichromophore as compared to directexcitation with incident light of an equivalent intensity. The signalmay be measured using any convenient method. In some cases, the 1.5× orgreater signal refers to an intensity of emitted light. In certaincases, the 1.5× or greater signal refers to an increased signal to noiseratio. In certain embodiments of the polymeric tandem dye, the signalingchromophore emission is 1.5 fold greater or more when excited by themultichromophore as compared to direct excitation of the signalingchromophore with incident light, such as 2-fold or greater, 3-fold orgreater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 8-foldor greater, 10-fold or greater, 20-fold or greater, 50-fold or greater,100-fold or greater, or even greater as compared to direct excitation ofthe signaling chromophore with incident light.

The linked luminescent signaling chromophore emission of the polymerictandem dye can have a quantum yield of 0.03 or more, such as a quantumyield of 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 ormore, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or moreor even more. In some instances, the polymeric tandem dye has anextinction coefficient of 5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹or more, 7×10⁵ cm⁻¹M⁻¹ or more, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ ormore, such as 1×10⁶ cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶cm⁻¹M⁻¹ or more, 2.5×10⁶ cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶cm⁻¹M⁻¹ or more, 5×10⁶ cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶cm⁻¹M⁻¹ or more, or 8×10⁶ cm⁻¹M⁻¹ or more. In some embodiments, thepolymeric tandem dye has a molar extinction coefficient of 5×10⁵ M⁻¹cm⁻¹ or more. In certain embodiments, the polymeric tandem dye has amolar extinction coefficient of 1×10⁶ M⁻¹ cm⁻¹ or more.

The subject polymeric tandem dyes provide for fluorescence emissionsfrom luminescent signaling chromophore dyes that are brighter than theemissions which are possible from such luminescent dyes in isolation.The linked luminescent signaling chromophore emission of the polymerictandem dye can have a brightness of 50 mM⁻¹ cm⁻¹ or more, such as 60mM⁻¹ cm⁻¹ or more, 70 mM⁻¹ cm⁻¹ or more, 80 mM⁻¹ cm⁻¹ or more, 90 mM⁻¹cm⁻¹ or more, 100 mM⁻¹ cm⁻¹ or more, 150 mM⁻¹ cm⁻¹ or more, 200 mM⁻¹cm⁻¹ or more, 250 mM⁻¹ cm⁻¹ or more, 300 mM⁻¹ cm⁻¹ or more, or evenmore. In certain instances, the linked signaling chromophore emission ofthe polymeric tandem dye has a brightness that is at least 5-foldgreater than the brightness of a directly excited luminescent dye, suchas at least 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 50-fold greater, at least 100-fold greater, at least300-fold greater, or even greater than the brightness of a directlyexcited luminescent dye.

In some embodiments, a polymeric tandem dye includes: a water solvatedpolymeric dye having a deep ultraviolet excitation spectrum andcomprising a segment of π-conjugated co-monomers and aconjugation-modifying repeat unit; and a signaling chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith.

In certain embodiments or the formulae described herein, e.g., formulae(I) and (III), a linked signaling chromophore (e.g., as describedherein) is included as a substituent of a co-monomer. Any convenientsignaling chromophore can be attached to any convenient polymer dyedescribed herein via coupling of compatible chemoselective functionalgroups. The signaling chromophore can be selected to provide for adesirable emission spectra and emission maximum wavelength.

As used herein, the terms “chemoselective functional group” and“chemoselective tag” are used interchangeably and refer to a functionalgroup that can selectively react with another compatible functionalgroup to form a covalent bond, in some cases, after optional activationof one of the functional groups. Chemoselective functional groups ofinterest include, but are not limited to, thiols and maleimide oriodoacetamide, amines and carboxylic acids or active esters thereof, aswell as groups that can react with one another via Click chemistry,e.g., azide and alkyne groups (e.g., cyclooctyne groups), as well ashydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne,phosphine, epoxide, and the like.

Any convenient linking co-monomers may be incorporated into the subjectmultichromophores to provide for a linking group to which may beattached any convenient moieties of interest (e.g., a linked signalingchromophore). Linking co-monomers of interest include, but are notlimited to, those co-monomers described in the formulae herein, and afluorene co-monomer, a phenylenevinylene co-monomer, aphenyleneethynylene co-monomer, a carbazole co-monomer, a C₂-C₁₂ alkyneco-monomer, an arylene-ethynylene co-monomer, a heteroarylene-ethynyleneco-monomer, an arylene co-monomer and a heteroarylene co-monomer. Asused herein, the terms aryl or heteroaryl co-monomer and arylene orheteroarylene co-monomer are used interchangeably. In certain cases, thelinking co-monomer is a substituted aryl co-monomer. In certain cases,the linking co-monomer is a substituted heteroaryl co-monomer. Incertain cases, the linking co-monomer is a substituted or unsubstituted1,4-phenyl, a substituted or unsubstituted 1,3-phenyl, a substituted orunsubstituted 4,4′-biphenyl, a substituted or unsubstituted 2,5-pyridyl,and a substituted or unsubstituted 2,6-pyridyl.

In some instances of any of the formula described herein, the signalingchromophore is linked to a co-monomer comprising 1% to 50% by molarityof the multichromophore, such as 1% to 20%, 1% to 10%, or 11 to 20% bymolarity. In certain cases, the multichromophore is a conjugated polymercomprising 5 or more repeat units.

Any convenient chemoselective functional groups may be included in thesubject multichromophores (e.g., at the —Z¹, —Z² and/or in the G¹ or G²terminal groups, including, but are not limited to, carboxylic acid,active ester (e.g., NHS or sulfo-NHS ester), amino, hydroxyl, thiol,maleimide, iodoacetyl, hydrazido, hydrazino, aldehyde, ketone, azido,alkyne, phosphine and epoxide. It is understood that in the polymerictandem dye structures described herein, in some cases, the groups Z¹ andZ² appear at a equivalent position in the structure where these groupscan be used interchangeably to refer to either a linked signalingchromophore or a chemoselective functional group that is capable ofsubsequent conjugation to a convenient chromophore precursor to producethe linked signaling chromophore.

In some cases, the signaling chromophore is a fluorophore. In certaincases, the signaling chromophore is a quencher. Any convenientfluorescent dyes may be utilized in the polymeric tandem dyes as anacceptor chromophore. The terms “fluorescent dye” and “fluorophore” areused interchangeably herein. The signaling chromophore (Z¹) can be a dyemolecule selected from a rhodamine, a coumarin, a xanthene, a cyanine, apolymethine, a pyrene, a dipyrromethene borondifluoride, a napthalimide,a phycobiliprotein, a peridinum chlorophyll protein, conjugates thereof,and combinations thereof. In certain embodiments, the signalingchromophore (Z¹) is a cyanine dye, a xanthene dye, a coumarin dye, athiazine dye and an acridine dye. In some instances, the signalingchromophore (Z¹) is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5 anddiethylamino coumarin. In some embodiments, the acceptor chromophore isa cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye or anacridine dye. Fluorescent dyes of interest include, but are not limitedto, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine,carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-aProtein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700,7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665, BODIPY R6G, BODIPY TMR, BODIPYTR, conjugates thereof, and combinations thereof. Lanthanide chelates ofinterest include, but are not limited to, europium chelates, terbiumchelates and samarium chelates. In some embodiments, the polymerictandem dye includes a polymeric dye linked to an acceptor fluorophoreselected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 andAlexa700. In certain embodiments, the polymeric tandem dye includes apolymeric dye linked to an acceptor fluorophore selected from Dyomicsdyes (such as DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), BiotiumCF 555, Cy 3.5, and diethylamino coumarin.

In some embodiments, the signaling chromophore that is selected has anemission maximum wavelength in the range of 300 to 900 nm, such as 350to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm,390 to 500 nm or 400 to 500 nm, where specific examples of emissionmaxima of signaling chromophore of interest include, but are not limitedto: 395 nm±5 nm, 420 nm±5 nm, 430 nm±5 nm, 440 nm±5 nm, 450 nm±5 nm, 460nm±5 nm, 470 nm±5 nm, 480 nm±5 nm, 490 nm±5 nm, 500 nm±5 nm, 510 nm±5nm, 520 nm±5 nm, 530 nm±5 nm, 540 nm±5 nm, 550 nm±5 nm, 560 nm±5 nm, 570nm±5 nm, 580 nm±5 nm, 590 nm±5 nm, 605 nm±5 nm, 650 nm±5 nm, 680 nm±5nm, 700 nm±5 nm, 805 nm±5 nm.

End Groups

Any convenient end groups (e.g., G¹ and G²) may be utilized at theterminals of the subject multichromophores. As used herein, the terms“end group” and “terminal group” are used interchangeably to refer tothe groups located at the terminals of the polymeric structure of themultichromophore, e.g., as described herein. G¹ and G² groups ofinterest include, but are not limited to a terminal capping group, a πconjugated segment, a linker and a linked specific binding member. Insome embodiments, a terminal capping groups is a monovalent group whichis conjugated to the backbone of the multichromophore afterpolymerization. In certain instances, the terminal capping group is anaryl, a substituted aryl, a heteroaryl, a substituted heteroaryl, analkyl or a substituted alkyl. In some cases, the terminal co-monomer isdirected linked to a chemoselective tag or linker. In certain cases, theterminal capping group is derived from a monomer used in the method ofpolymerization, e.g., a terminal group such as a halogen (e.g., Br), aboronic acid or a boronic ester, which is capable of undergoing furtherconjugation. In some instances, G¹ and/or G² is a π conjugated segment.As used herein, a π conjugated segment refers to any convenient segmentof a conjugated polymer to which the multichromophore may be conjugated,i.e., allowing delocalization of pi electron across adjacent units. Incertain embodiments, G¹ and/or G² is a linker, such as a linkerincluding a functional group suitable for conjugation to a specificbinding moiety. It is understood that linkers located at the G¹ and/orG² positions of the multichromophore may be selected so as to beorthogonal to any other linkers including chemoselective tags (e.g., asdescribed herein) that may be present at a sidechain of themultichromophore (e.g., at Z²). In certain embodiments, an aminofunctional group or derivative thereof is included at G¹ and/or G² and acarboxylic acid functional group or derivative thereof is included atZ². In certain embodiments, a carboxylic acid functional group orderivative thereof is included at G¹ and/or G² and an amino functionalgroup or derivative thereof is included at Z².

In some embodiments of the formulae described herein, at least one of G¹and G² is -L³-Z⁴ where L³ is a linker (e.g., as described herein) and Z⁴is a specific binding member (e.g., as described herein). In someembodiments of formulae described herein, at least one of G¹ and G² is-L³-Z³ where L³ is a linker (e.g., as described herein) and Z³ is achemoselective tag (e.g., as described herein). Any convenientchemoselective tag and conjugation chemistries can be adapted for use inthe subject multichromophores. Chemoselective tags of interest include,but are not limited to, amine, active ester, maleimide, thiol,sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, DiersAlder cycloaddition click reagents and click chemistry, tetrazine,transcyclooctene, aldehyde, alkoxylamine, alkynes, cyclooctynes, azide,and the like. In some instances, Z³ is selected from the groupconsisting of carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl andthiol. In certain embodiments of formulae described herein, at least oneof G¹ and G² is described by the following structure:*—Ar-L-Z

where Ar is a π-conjugated aryl group, L is a linker and Z is achemoselective tag or a specific binding member. In some cases, the L-Zgroup can be connected directed to a terminal co-monomer. In certainembodiments of formulae described herein, at least one of G¹ and G² isdescribed by the following structure:

wherein:

q is 0 or an integer from 1-12;

L is an optional linker; and

Z is a chemoselective tag or a specific binding member. In certainembodiments, Z is a biomolecule. Biomolecules of interest include, butare not limited to, polypeptides, polynucleotides, carbohydrates, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsthereof and combinations thereof. In certain instances, Z is anantibody. In some instances, Z is an antibody fragment or bindingderivative thereof. In some cases, the antibody fragment or bindingderivative thereof is selected from the group consisting of a Fabfragment, a F(ab′)₂ fragment, a scFv, a diabody and a triabody.

It is understood that for any of the structures and formula depictedherein that in some cases of the subject multichromophore the end groupsdepicted may be located at the opposite ends to those shown, e.g., theend groups G¹ and -Ph-L-Z may be switched. In some embodiments of themultichromophores described herein (e.g., formulae (II) to (V)), atleast one of G¹ and G² is selected from one of the following structures1-33:

-   *=site for covalent attachment to unsaturated backbone;-   wherein R′ is independently H, halogen, C₁-C₁₂alkyl,    (C₁-C₁₂alkyl)NH₂, C₂-C₁₂alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl,    C₁-C₁₂haloalkyl, C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino,    —[CH₂—CH₂]_(r)—Z¹, or (C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or    —COOH; X¹ is —NH₂, —NHCOOH, —NHCOOC(CH₃)₃,    —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or    —NHCO[CH₂—CH₂—O]_(s′)(CH₂)_(s′)NH₂; r′ is an integer from 1 to 20;    and each s′ is independently an integer from 1 to 20,-   (CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0    to 50, or a benzyl optionally substituted with one or more halogen,    hydroxyl, C₁-C₁₂ alkoxy, or-   (OCH₂CH₂)_(y″)CH₃ where each y″ is independently an integer from 0    to 50 and R′ is different from R;-   wherein k is 2, 4, 8, 12 or 24;-   wherein R¹⁵ is selected from the group consisting of I-u having the    structure:

*=site for covalent attachment to backbone.

In some embodiments of the multichromophores described herein (e.g.,formulae (I)-(V), at least one end group (e.g., T²-Z², G¹, G², -L-Z,-L³-Z³), or sidechain group, is selected from one of the followingstructures:

wherein r is 0 or an integer from 1-50; k is 0 or an integer from 1-50(e.g., 1-20); R¹ is as defined for any of the fluorene co-monomersdescribed herein; and R¹⁶ is selected from H, OH, NH₂, —NH(CH₂)_(r)—NH₂,and —NH(CH₂)_(r)COOH. In certain instances, r is 1 to 20, such as 3 to20, 3 to 15, 3 to 12, or 6 to 12. In certain cases, r is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12. In some cases, r is 3. In some cases, r is 4.In some cases, r is 5. In some cases, r is 6. In some cases, r is 7. Insome cases, r is 8. In some cases, r is 9. In some cases, r is 10. Insome cases, r is 11.

In some embodiments, the multichromophore includes one or more of thefollowing groups, e.g., as an end group or sidechain group forconjugation to a compatible functional group on another molecule:

Labelled Specific Binding Members

Aspects of the present disclosure include labelled specific bindingmembers. A labelled specific binding member is a conjugate of a subjectpolymeric dye (e.g., as described herein) and a specific binding member.Any of the polymeric dyes or polymeric tandem dyes described herein maybe conjugated to a specific binding member. The specific binding memberand the polymeric dye can be conjugated (e.g., covalently linked) toeach other at any convenient locations of the two molecules, via anoptional linker. In some embodiments, the labelled specific bindingmember is aggregation resistant. As used herein, by“aggregation-resistant” is meant a labelled specific binding membercapable of forming a homogenous aqueous composition without aggregatedprecipitate at a concentration of 1 mg/ml or more in an aqueous bufferof interest, such as 2 mg/ml or more, 3 mg/ml or more, 4 mg/ml or more,5 mg/ml or more, 6 mg/ml or more, 7 mg/ml or more, 8 mg/ml or more, 9mg/ml or more, 10 mg/mL or more or even more of the labelled specificbinding member.

In certain embodiments, the labelled specific binding member comprises:a water solvated polymeric dye having a deep ultraviolet excitationspectrum and comprising a segment of π-conjugated co-monomers and aconjugation-modifying repeat unit; and a specific binding membercovalently linked to the multichromophore.

As used herein, the term “specific binding member” refers to one memberof a pair of molecules which have binding specificity for one another.One member of the pair of molecules may have an area on its surface, ora cavity, which specifically binds to an area on the surface of, or acavity in, the other member of the pair of molecules. Thus the membersof the pair have the property of binding specifically to each other toproduce a binding complex. In some embodiments, the affinity betweenspecific binding members in a binding complex is characterized by aK_(d) (dissociation constant) of 10⁻⁶ M or less, such as 10⁻⁷ M or less,including 10⁻⁸ M or less, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, including10⁻¹⁵ M or less. In some embodiments, the specific binding membersspecifically bind with high avidity. By high avidity is meant that thebinding member specifically binds with an apparent affinitycharacterized by an apparent K_(d) of 10×10⁻⁹ M or less, such as 1×10⁻⁹M or less, 3×10⁻¹⁰ M or less, 1×10⁻¹⁰ M or less, 3×10⁻¹¹ M or less,1×10⁻¹¹ M or less, 3×10⁻¹² M or less or 1×10⁻¹² M or less.

The specific binding member can be proteinaceous. As used herein, theterm “proteinaceous” refers to a moiety that is composed of amino acidresidues. A proteinaceous moiety can be a polypeptide. In certain cases,the proteinaceous specific binding member is an antibody. In certainembodiments, the proteinaceous specific binding member is an antibodyfragment, e.g., a binding fragment of an antibody that specific binds toa polymeric dye. As used herein, the terms “antibody” and “antibodymolecule” are used interchangeably and refer to a protein consisting ofone or more polypeptides substantially encoded by all or part of therecognized immunoglobulin genes. The recognized immunoglobulin genes,for example in humans, include the kappa (k), lambda (l), and heavychain genetic loci, which together comprise the myriad variable regiongenes, and the constant region genes mu (u), delta (d), gamma (g), sigma(e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. An immunoglobulin light or heavy chain variable regionconsists of a “framework” region (FR) interrupted by three hypervariableregions, also called “complementarity determining regions” or “CDRs”.The extent of the framework region and CDRs have been precisely defined(see, “Sequences of Proteins of Immunological Interest,” E. Kabat etal., U.S. Department of Health and Human Services, (1991)). Thenumbering of all antibody amino acid sequences discussed herein conformsto the Kabat system. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs. The CDRs are primarily responsible for binding to an epitopeof an antigen. The term antibody is meant to include full lengthantibodies and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.

Antibody fragments of interest include, but are not limited to, Fab,Fab′, F(ab′)₂, Fv, scFv, or other antigen-binding subsequences ofantibodies, either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies. Antibodiesmay be monoclonal or polyclonal and may have other specific activitieson cells (e.g., antagonists, agonists, neutralizing, inhibitory, orstimulatory antibodies). It is understood that the antibodies may haveadditional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other antibody functions.

In certain embodiments, the specific binding member is a Fab fragment, aF(ab′)₂ fragment, a scFv, a diabody or a triabody. In certainembodiments, the specific binding member is an antibody. In some cases,the specific binding member is a murine antibody or binding fragmentthereof. In certain instances, the specific binding member is arecombinant antibody or binding fragment thereof.

In some embodiments, the labelled specific binding member includes: awater solvated light harvesting multichromophore (e.g., as describedherein); and a signaling chromophore covalently linked to themultichromophore in energy-receiving proximity therewith (e.g., asdescribed herein); and a specific binding member covalently linked tothe multichromophore. In some instances of the labelled specific bindingmember, the multichromophore of any of the formula described herein(e.g., formulae (I)-(V)), wherein: G¹ and G² are each independentlyselected from the group consisting of a terminal group (e.g., endgroup), a π conjugated segment, a linker and a linked specific bindingmember, wherein at least one of G¹ and G² is a linked specific bindingmember.

Methods

As summarized above, aspects of the invention include methods ofevaluating a sample for the presence of a target analyte. Aspects of themethod include contacting the sample with a polymeric dye conjugate thatspecifically binds the target analyte to produce a labelling compositioncontacted sample. As used herein, the terms “polymeric dye conjugate”and “labelled specific binding member” are used interchangeably. Assuch, the polymeric dye conjugate can include: (i) a water solvatedpolymeric dye (e.g., as described herein); and (ii) a specific bindingmember (e.g., as described herein). In some instances, the polymeric dyeconjugate further comprises a signaling chromophore covalently linked toa multichromophore of the polymeric dye in energy-receiving proximitytherewith.

Any convenient method may be used to contact the sample with a polymericdye conjugate that specifically binds to the target analyte to producethe labelling composition contacted sample. In some instances, thesample is contacted with the polymeric dye conjugate under conditions inwhich the specific binding member specifically binds to the targetanalyte, if present. For specific binding of the specific binding memberof the conjugate with the target analyte, an appropriate solution may beused that maintains the biological activity of the components of thesample and the specific binding member. The solution may be a balancedsalt solution, e.g., normal saline, PBS, Hank's balanced salt solution,etc., conveniently supplemented with fetal calf serum, human plateletlysate or other factors, in conjunction with an acceptable buffer at lowconcentration, such as from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc. Various media are commerciallyavailable and may be used according to the nature of the target analyte,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., in some casessupplemented with fetal calf serum or human platelet lysate. The finalcomponents of the solution may be selected depending on the componentsof the sample which are included.

The temperature at which specific binding of the specific binding memberof the conjugate to the target analyte takes place may vary, and in someinstances may range from 5° C. to 50° C., such as from 10° C. to 40° C.,15° C. to 40° C., 20° C. to 40° C., e.g., 20° C., 25° C., 30° C., 35° C.or 37° C. (e.g., as described above). In some instances, the temperatureat which specific binding takes place is selected to be compatible withthe biological activity of the specific binding member and/or the targetanalyte. In certain instances, the temperature is 25° C., 30° C., 35° C.or 37° C. In certain cases, the specific binding member is an antibodyor fragment thereof and the temperature at which specific binding takesplace is room temperature (e.g., 25° C.), 30° C., 35° C. or 37° C. Anyconvenient incubation time for specific binding may be selected to allowfor the formation of a desirable amount of binding complex, and in someinstances, may be 1 minute (min) or more, such as 2 min or more, 10 minor more, 30 min or more, 1 hour or more, 2 hours or more, or even 6hours or more.

Any convenient specific binding members may be utilized in theconjugate. Specific binding members of interest include, but are notlimited to, those agents that specifically bind cell surface proteins ofa variety of cell types, including but not limited to, stem cells, e.g.,pluripotent stem cells, hematopoietic stem cells, T cells, T regulatorcells, dendritic cells, B Cells, e.g., memory B cells, antigen specificB cells, granulocytes, leukemia cells, lymphoma cells, virus cells(e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts,epithelial cells, endothelial cells, and erythroid cells. Target cellsof interest include cells that have a convenient cell surface marker orantigen that may be captured by a convenient specific binding memberconjugate. In some embodiments, the target cell is selected from HIVcontaining cell, a Treg cell, an antigen-specific T-cell populations,tumor cells or hematopoetic progenitor cells (CD34+) from whole blood,bone marrow or cord blood. Any convenient cell surface proteins or cellmarkers may be targeted for specific binding to polymeric dye conjugatesin the subject methods. In some embodiments, the target cell includes acell surface marker selected from a cell receptor and a cell surfaceantigen. In some cases, the target cell may include a cell surfaceantigen such as CD11 b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25,CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34,CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptorgamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5(N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.

Any convenient targets may be selected for evaluation utilizing thesubject methods. Targets of interest include, but are not limited to, anucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, aprotein, such as a fusion protein, a modified protein, such as aphosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylatedprotein, or an antibody, a peptide, an aggregated biomolecule, a cell, asmall molecule, a vitamin and a drug molecule. As used herein, the term“a target protein” refers to all members of the target family, andfragments thereof. The target protein may be any protein of interest,such as a therapeutic or diagnostic target, including but not limitedto: hormones, growth factors, receptors, enzymes, cytokines,osteoinductive factors, colony stimulating factors and immunoglobulins.The term “target protein” is intended to include recombinant andsynthetic molecules, which can be prepared using any convenientrecombinant expression methods or using any convenient syntheticmethods, or purchased commercially. In some embodiments, the polymericdye conjugates include an antibody or antibody fragment. Any convenienttarget analyte that specifically binds an antibody or antibody fragmentof interest may be targeted in the subject methods.

In some embodiments, the target analyte is associated with a cell. Incertain instances, the target analyte is a cell surface marker of thecell. In certain cases, the cell surface marker is selected from thegroup consisting of a cell receptor and a cell surface antigen. In someinstances, the target analyte is an intracellular target, and the methodfurther includes lysing the cell.

In some embodiments, the sample may include a heterogeneous cellpopulation from which target cells are isolated. In some instances, thesample includes peripheral whole blood, peripheral whole blood in whicherythrocytes have been lysed prior to cell isolation, cord blood, bonemarrow, density gradient-purified peripheral blood mononuclear cells orhomogenized tissue. In some cases, the sample includes hematopoeticprogenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cordblood. In certain embodiments, the sample includes tumor cells inperipheral blood. In certain instances, the sample is a sample including(or suspected of including) viral cells (e.g., HIV).

The labelled specific binding members find use in the subject methods,e.g., for labeling a target cell, particle, target or analyte with apolymeric dye or polymeric tandem dye. For example, labelled specificbinding members find use in labeling cells to be processed (e.g.,detected, analyzed, and/or sorted) in a flow cytometer. The labelledspecific binding members may include antibodies that specifically bindto, e.g., cell surface proteins of a variety of cell types (e.g., asdescribed herein). The labelled specific binding members may be used toinvestigate a variety of biological (e.g., cellular) properties orprocesses such as cell cycle, cell proliferation, cell differentiation,DNA repair, T cell signaling, apoptosis, cell surface protein expressionand/or presentation, and so forth. Labelled specific binding members maybe used in any application that includes (or may include)antibody-mediated labeling of a cell, particle or analyte.

In some instances of the method, the labelled specific binding memberincludes a multichromophore as described herein (eg., according to anyone of formulae (I)-(IV)). In certain cases, G¹ and G² are eachindependently selected from the group consisting of a terminal group, aπ conjugated segment, a linker and a linked specific binding member,wherein at least one of G¹ and G² is a linked specific binding member.

Aspects of the method include assaying the labelling compositioncontacted sample for the presence of a polymeric dye conjugate-targetanalyte binding complex to evaluate whether the target analyte ispresent in the sample. Once the sample has been contacted with thepolymeric dye conjugate, any convenient methods may be utilized inassaying the labelling composition contacted sample that is produced forthe presence of a polymeric dye conjugate-target analyte bindingcomplex. The polymeric dye conjugate-target analyte binding complex isthe binding complex that is produced upon specific binding of thespecific binding member of the conjugate to the target analyte, ifpresent. Assaying the labelling composition contacted sample can includedetecting a fluorescent signal from the binding complex, if present. Insome cases, the assaying includes a separating step where the targetanalyte, if present, is separated from the sample. A variety of methodscan be utilized to separate a target analyte from a sample, e.g., viaimmobilization on a support. Assay methods of interest include, but arenot limited to, any convenient methods and assay formats where pairs ofspecific binding members such as avidin-biotin or hapten-anti-haptenantibodies find use, are of interest. Methods and assay formats ofinterest that may be adapted for use with the subject compositionsinclude, but are not limited to, flow cytometry methods, in-situhybridization methods, enzyme-linked immunosorbent assays (ELISAs),western blot analysis, magnetic cell separation assays and fluorochromepurification chromatography.

In certain embodiments, the method further includes contacting thesample with a second specific binding member that specifically binds thetarget analyte. In certain instances, the second specific binding memberis support bound. Any convenient supports may be utilized to immobilizea component of the subject methods (e.g., a second specific bindingmember). In certain instances, the support is a particle, such as amagnetic particle. In some instances, the second specific binding memberand the polymeric dye conjugate produce a sandwich complex that may beisolated and detected, if present, using any convenient methods. In someembodiments, the method further includes flow cytometrically analyzingthe polymeric dye conjugate-target analyte binding complex, i.e., afluorescently labelled target analyte. Assaying for the presence of apolymeric dye conjugate-target analyte binding complex may provide assayresults (e.g., qualitative or quantitative assay data) which can be usedto evaluate whether the target analyte is present in the sample.

Any convenient supports may be utilized in the subject methods toimmobilize any convenient component of the methods, e.g., labelledspecific binding member, target, secondary specific binding member, etc.Supports of interest include, but are not limited to: solid substrates,where the substrate can have a variety of configurations, e.g., a sheet,bead, or other structure, such as a plate with wells; beads, polymers,particle, a fibrous mesh, hydrogels, porous matrix, a pin, a microarraysurface, a chromatography support, and the like. In some instances, thesupport is selected from the group consisting of a particle, a planarsolid substrate, a fibrous mesh, a hydrogel, a porous matrix, a pin, amicroarray surface and a chromatography support. The support may beincorporated into a system that it provides for cell isolation assistedby any convenient methods, such as a manually-operated syringe, acentrifuge or an automated liquid handling system. In some cases, thesupport finds use in an automated liquid handling system for the highthroughput isolation of cells, such as a flow cytometer.

In some embodiments of the method, the separating step includes applyingan external magnetic field to immobilize a magnetic particle. Anyconvenient magnet may be used as a source of the external magnetic field(e.g., magnetic field gradient). In some cases, the external magneticfield is generated by a magnetic source, e.g. by a permanent magnet orelectromagnet. In some cases, immobilizing the magnetic particles meansthe magnetic particles accumulate near the surface closest to themagnetic field gradient source, i.e. the magnet.

The separating may further include one or more optional washing steps toremove unbound material of the sample from the support. Any convenientwashing methods may be used, e.g., washing the immobilized support witha biocompatible buffer which preserves the specific binding interactionof the polymeric dye and the specific binding member. Separation andoptional washing of unbound material of the sample from the supportprovides for an enriched population of target cells where undesiredcells and material may be removed.

In certain embodiments, the method further includes detecting thelabelled target. Detecting the labelled target may include exciting themultichromophore with one or more lasers and subsequently detectingfluorescence emission from the polymeric tandem dye using one or moreoptical detectors. Detection of the labelled target can be performedusing any convenient instruments and methods, including but not limitedto, flow cytometry, FACS systems, fluorescence microscopy; fluorescence,luminescence, ultraviolet, and/or visible light detection using a platereader; high performance liquid chromatography (HPLC); and massspectrometry. When using fluorescently labeled components in the methodsand compositions of the present disclosure, it is recognized thatdifferent types of fluorescence detection systems can be used topractice the subject methods. In some cases, high throughput screeningcan be performed, e.g., systems that use 96 well or greater microtiterplates. A variety of methods of performing assays on fluorescentmaterials can be utilized, such as those methods described in, e.g.,Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B., Resonance energy transfer microscopy,in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methodsin Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego:Academic Press (1989), pp. 219-243; Turro, N. J., Modern MolecularPhotochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.(1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. In somecases, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescently labelled targets in the sample emit radiation which has awavelength that is different from the excitation wavelength. Collectionoptics then collect the emission from the sample. The device can includea temperature controller to maintain the sample at a specifictemperature while it is being scanned. In certain instances, amulti-axis translation stage moves a microtiter plate holding aplurality of samples in order to position different wells to be exposed.The multi-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

In some embodiments, the method of evaluating a sample for the presenceof a target analyte further includes detecting fluorescence in a flowcytometer. In some embodiments, the method of evaluating a sample forthe presence of a target analyte further includes imaging the labellingcomposition contacted sample using fluorescence microscopy. Fluorescencemicroscopy imaging can be used to identify a polymeric dyeconjugate-target analyte binding complex in the contacted sample toevaluate whether the target analyte is present. Microscopy methods ofinterest that find use in the subject methods include laser scanningconfocal microscopy.

Also provided are methods of labelling a target molecule. The subjectpolymeric dyes, find use in a variety of methods of labelling,separation, detection and/or analysis. In some embodiments, the methodincludes: contacting the target molecule with a polymeric dye (e.g., asdescribed herein) to produce a labelled target molecule, wherein thepolymeric dye includes a conjugation tag that covalently links to thetarget molecule. In some cases, the polymeric dye further includes asignaling chromophore covalently linked to the multichromophore of thepolymeric dye in energy-receiving proximity therewith. In some instancesof the method, the polymeric dye member includes a multichromophoreaccording to any one of formulae (I)-(IV) (e.g., as described herein),where one of G¹ and G² is a terminal group and the other of G¹ and G² isthe conjugation tag.

As used herein the term “conjugation tag” refers to a group thatincludes a chemoselective functional group (e.g., as described herein)that can covalently link with a compatible functional group of a targetmolecule, after optional activation and/or deprotection. Any convenientconjugation tags may be utilized in the subject polymeric dyes in orderto conjugate the dye to a target molecule of interest. In someembodiments, the conjugation tag includes a terminal functional groupselected from an amino, a carboxylic acid or a derivative thereof, athiol, a hydroxyl, a hydrazine, a hydrazide, a azide, an alkyne and aprotein reactive group (e.g. amino-reactive, thiol-reactive,hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive).

Any convenient methods and reagent may be adapted for use in the subjectlabelling methods in order to covalently link the conjugation tag to thetarget molecule. Methods of interest for labelling a target, include butare not limited to, those methods and reagents described by Hermanson,Bioconjugate Techniques, Third edition, Academic Press, 2013. Thecontacting step may be performed in an aqueous solution. In someinstances, the conjugation tag includes an amino functional group andthe target molecule includes an activated ester functional group, suchas a NHS ester or sulfo-NHS ester, or vice versa. In certain instances,the conjugation tag includes a maleimide functional group and the targetmolecule includes a thiol functional group, or vice versa. In certaininstances, the conjugation tag includes an alkyne (e.g., a cyclooctynegroup) functional group and the target molecule includes an azidefunctional group, or vice versa, which can be conjugated via Clickchemistry.

Any convenient target molecules may be selected for labelling utilizingthe subject methods. Target molecules of interest include, but are notlimited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA orANA molecule, a protein, such as a fusion protein, a modified protein,such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, oracetylated protein, or an antibody, a peptide, an aggregatedbiomolecule, a cell, a small molecule, a vitamin and a drug molecule. Asused herein, the term “a target protein” refers to all members of thetarget family, and fragments thereof. The target protein may be anyprotein of interest, such as a therapeutic or diagnostic target,including but not limited to: hormones, growth factors, receptors,enzymes, cytokines, osteoinductive factors, colony stimulating factorsand immunoglobulins. The term “target protein” is intended to includerecombinant and synthetic molecules, which can be prepared using anyconvenient recombinant expression methods or using any convenientsynthetic methods, or purchased commercially. In some embodiments, thetarget molecule is a specific binding member (e.g., as describedherein). In certain instances, the specific binding member is anantibody. In some instances, the specific binding member is an antibodyfragment or binding derivative thereof. In some case, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody.

In some cases, the method includes a separating step where the labelledtarget molecule is separated from the reaction mixture, e.g., excessreagents or unlabeled target. A variety of methods may be utilized toseparate a target from a sample, e.g., via immobilization on a support,precipitation, chromatography, and the like.

In some instances, the method further includes detecting and/oranalyzing the labelled target molecule. In some instances, the methodfurther includes fluorescently detecting the labelled target molecule.Any convenient methods may be utilized to detect and/or analyze thelabelled target molecule in conjunction with the subject methods andcompositions. Methods of analyzing a target of interest that find use inthe subject methods, include but are not limited to, flow cytometry,fluorescence microscopy, in-situ hybridization, enzyme-linkedimmunosorbent assays (ELISAs), western blot analysis, magnetic cellseparation assays and fluorochrome purification chromatography.Detection methods of interest include but are not limited tofluorescence spectroscopy, fluorescence microscopy, nucleic acidsequencing, fluorescence in-situ hybridization (FISH), protein massspectroscopy, flow cytometry, and the like.

Detection may be achieved directly via the polymeric dye or polymerictandem dye, or indirectly by a secondary detection system. The lattermay be based on any one or a combination of several different principlesincluding, but not limited to, antibody labelled anti-species antibodyand other forms of immunological or non-immunological bridging andsignal amplification systems (e.g., biotin-streptavidin technology,protein-A and protein-G mediated technology, or nucleic acidprobe/anti-nucleic acid probes, and the like). Suitable reportermolecules may be those known in the field of immunocytochemistry,molecular biology, light, fluorescence, and electron microscopy, cellimmunophenotyping, cell sorting, flow cytometry, cell visualization,detection, enumeration, and/or signal output quantification. More thanone antibody of specific and/or non-specific nature might be labelledand used simultaneously or sequentially to enhance target detection,identification, and/or analysis.

Systems

Aspects of the invention further include systems for use in practicingthe subject methods and compositions. A sample analysis system caninclude sample field of view or a flow channel loaded with a sample anda labelled specific binding member. In some embodiments, the system is aflow cytometric system including: a flow cytometer including a flowpath; a composition in the flow path, wherein the composition includes:a sample; and a labelled specific binding member (e.g., as describedherein).

In some embodiments, the system for analyzing a sample is a fluorescencemicroscopy system, including: a fluorescence microscope comprising asample field of view; and a composition disposed in the sample field ofview, wherein the composition comprises a sample; and a labelledspecific binding member (e.g., as described herein).

In some instances of the systems, the labelled specific binding memberincludes: a water solvated light harvesting multichromophore (e.g., asdescribed herein) and a specific binding member that specifically bindsa target analyte covalently linked to the multichromophore. In somecases, the labelled specific binding member further comprises asignaling chromophore covalently linked to the multichromophore of thepolymeric dye in energy-receiving proximity therewith. In some instancesof the subject systems, the labelled specific binding member, themultichromophore is described by any one of formulae (I)-(IV) (e.g., asdescribed herein), wherein: G¹ and G² are each independently selectedfrom the group consisting of a terminal group, a π conjugated segment, alinker and a linked specific binding member, wherein at least one of G¹and G² is a linked specific binding member.

In certain embodiments of the systems, the composition further includesa second specific binding member that is support bound and specificallybinds the target analyte. In some cases, the support includes a magneticparticle. As such, in certain instances, the system may also include acontrollable external paramagnetic field configured for application toan assay region of the flow channel.

The sample may include a cell. In some instances, the sample is acell-containing biological sample. In some instances, the sampleincludes a labelled specific binding member specifically bound to atarget cell. In certain instances, the target analyte that isspecifically bound by the specific binding member is a cell surfacemarker of the cell. In certain cases, the cell surface marker isselected from the group consisting of a cell receptor and a cell surfaceantigen.

In certain aspects, the system may also include a light sourceconfigured to direct light to an assay region of the flow channel orsample field of view. The system may include a detector configured toreceive a signal from an assay region of the flow channel or a samplefield of view, wherein the signal is provided by the fluorescentcomposition. Optionally further, the sample analysis system may includeone or more additional detectors and/or light sources for the detectionof one or more additional signals.

In certain aspects, the system may further include computer-basedsystems configured to detect the presence of the fluorescent signal. A“computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of the computer-based systems of thepresent invention includes a central processing unit (CPU), input means,output means, and data storage means. A skilled artisan can readilyappreciate that any one of the currently available computer-based systemare suitable for use in the subject systems. The data storage means mayinclude any manufacture including a recording of the present informationas described above, or a memory access means that can access such amanufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g., word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In certain aspects, the system includes a flow cytometer. Flowcytometers of interest include, but are not limited, to those devicesdescribed in U.S. Pat. Nos. 4,704,891; 4,727,029; 4,745,285; 4,867,908;5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791;the disclosures of which are herein incorporated by reference.

Other systems may find use in practicing the subject methods. In certainaspects, the system may be a fluorimeter or microscope loaded with asample having a fluorescent composition of any of the embodimentsdiscussed herein. The fluorimeter or microscope may include a lightsource configured to direct light to the assay region of the flowchannel or sample field of view. The fluorimeter or microscope may alsoinclude a detector configured to receive a signal from an assay regionof the flow channel or field of view, wherein the signal is provided bythe fluorescent composition.

Kits

Aspects of the invention further include kits for use in practicing thesubject methods and compositions. The compositions of the invention canbe included as reagents in kits either as starting materials or providedfor use in, for example, the methodologies described above.

A kit can include a polymeric dye including a water solvated lightharvesting multichromophore (e.g., as described herein) and a container.Any convenient containers can be utilized, such as tubes, bottles, orwells in a multi-well strip or plate, a box, a bag, an insulatedcontainer, and the like. The subject kits can further include one ormore components selected from a polymeric tandem dye, a fluorophore, aspecific binding member, a specific binding member conjugate, a supportbound specific binding member, a cell, a support, a biocompatibleaqueous elution buffer, and instructions for use. In some embodiments ofthe kit, the multichromophore is covalently linked to a specific bindingmember. In some instances, the specific binding member is an antibody.In certain instances, the specific binding member is an antibodyfragment or binding derivative thereof. In certain cases, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody.

In certain embodiments, the kit finds use in evaluating a sample for thepresence of a target analyte, such as an intracellular target. As such,in some instances, the kit includes one or more components suitable forlysing cells. The one or more additional components of the kit may beprovided in separate containers (e.g., separate tubes, bottles, or wellsin a multi-well strip or plate).

In certain aspects, the kit further includes reagents for performing aflow cytometric assay. Reagents of interest include, but are not limitedto, buffers for reconstitution and dilution, buffers for contacting acell sample the multichromophore, wash buffers, control cells, controlbeads, fluorescent beads for flow cytometer calibration and combinationsthereof. The kit may also include one or more cell fixing reagents suchas paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or anycombinations or buffers thereof. Further, the kit may include a cellpermeabilizing reagent, such as methanol, acetone or a detergent (e.g.,triton, NP-40, saponin, tween 20, digitonin, leucoperm, or anycombinations or buffers thereof. Other protein transport inhibitors,cell fixing reagents and cell permeabilizing reagents familiar to theskilled artisan are within the scope of the subject kits.

The compositions of the kit may be provided in a liquid composition,such as any suitable buffer. Alternatively, the compositions of the kitmay be provided in a dry composition (e.g., may be lyophilized), and thekit may optionally include one or more buffers for reconstituting thedry composition. In certain aspects, the kit may include aliquots of thecompositions provided in separate containers (e.g., separate tubes,bottles, or wells in a multi-well strip or plate).

In addition, one or more components may be combined into a singlecontainer, e.g., a glass or plastic vial, tube or bottle. In certaininstances, the kit may further include a container (e.g., such as a box,a bag, an insulated container, a bottle, tube, etc.) in which all of thecomponents (and their separate containers) are present. The kit mayfurther include packaging that is separate from or attached to the kitcontainer and upon which is printed information about the kit, thecomponents of the and/or instructions for use of the kit.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, DVD, portable flash drive, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the Internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Utility

The polymeric dyes, compositions, methods and systems as describedherein may find use in a variety of applications, including diagnosticand research applications, in which the labelling, detection and/oranalysis of a target of interest is desirable. Such applications includemethodologies such as cytometry, microscopy, immunoassays (e.g.competitive or non-competitive), assessment of a free analyte,assessment of receptor bound ligand, and so forth. The compositions,system and methods described herein may be useful in analysis of any ofa number of samples, including but not limited to, biological fluids,cell culture samples, and tissue samples. In certain aspects, thecompositions, system and methods described herein may find use inmethods where analytes are detected in a sample, if present, usingfluorescent labels, such as in fluorescent activated cell sorting oranalysis, immunoassays, immunostaining, and the like. In certaininstances, the compositions and methods find use in applications wherethe evaluation of a sample for the presence of a target analyte is ofinterest.

In some cases, the methods and compositions find use in any assay formatwhere the detection and/or analysis of a target from a sample is ofinterest, including but not limited to, flow cytometry, fluorescencemicroscopy, in-situ hybridization, enzyme-linked immunosorbent assays(ELISAs), western blot analysis, magnetic cell separation assays andfluorochrome purification chromatography. In certain instances, themethods and compositions find use in any application where thefluorescent labelling of a target molecule is of interest. The subjectcompositions may be adapted for use in any convenient applications wherepairs of specific binding members find use, such as biotin-streptavidinand hapten-anti-hapten antibody.

ADDITIONAL EMBODIMENTS

Notwithstanding the appended claims, the disclosure set forth herein isalso defined by the following clauses:

-   Clause 1. A water solvated polymeric dye comprising:

a segment of π-conjugated co-monomers; and

a conjugation-modifying repeat unit;

wherein the polymeric dye has a deep ultraviolet excitation spectrum.

-   Clause 2. The polymeric dye according to clause 1, wherein the    polymeric dye comprises a plurality of π-conjugated segments    separated by conjugation-modifying repeat units.-   Clause 3. The polymeric dye according to clause 2, wherein the    plurality of π-conjugated segments each have a conjugation length of    20 or less.-   Clause 4. The polymeric dye according to any one of clauses 1-3,    wherein the excitation spectrum of the polymeric dye has a λ_(max)    of 350 nm or less.-   Clause 5. The polymeric dye according to any one of clauses 1-4,    wherein the conjugation-modifying repeat unit comprises the    structure:

wherein:

Z is selected from a covalent bond, a saturated atom, a saturated groupand a functional group, with the proviso that when Z is a covalent bondat least two of R²², R²³, R²⁵ and R²⁸ are not H (e.g., at least one ofR²² and R²³, and at least one of R²⁵ and R²⁸ is not H);

R²¹-R²⁸ are independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide,substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide, and a water solubilizing group, and wherein one or more ofR²², R²³, R²⁵ and R²⁸ is optionally cyclically linked to Z to provide abenzo-fused carbocycle or heterocycle ring; and

each * is a site for covalent attachment to the backbone of thepolymeric dye.

-   Clause 6. The polymeric dye according to clause 5, wherein Z is    selected from a saturated carbon atom, a saturated heteroatom, a    functional group and a saturated linker.-   Clause 7. The polymeric dye according to any one of clauses 5-6,    wherein:

Z is selected from —S—, —S(O)—, —SO₂—, —SO₂NR′—, —S(O)O—, —O—, —C(O)—,—C(O)NR′—, —C(O)O—, —OC(O)NR′—, —OC(O)O—, —NR′C(O)NR′—, —C(R⁵)(R⁶)—,—Si(R⁵)(R⁶)—, —N(R′)—, an alkyl linker and a polyethylene glycol linker;and

each R′, R⁵ and R⁶ is independently selected from H, alkyl, substitutedalkyl, or wherein R⁵ and/or R⁶ is optionally cyclically linked to R²²,R²³, R²⁵ or R²⁸ provide the benzo-fused carbocycle or heterocycle ring.

-   Clause 8. The polymeric dye according to clause 7, wherein Z is    selected from —S—, —S(O)—, —SO₂—, —O—, —C(O)—, —C(O)NR′—,    —OC(O)NR′—, —OC(O)O— and —NR′C(O)NR′—.-   Clause 9. The polymeric dye according to clause 7, wherein Z is    C(R⁵)(R⁶)— or —Si(R⁵)(R⁶)—.-   Clause 10. The polymeric dye according to clause 9, wherein the    conjugation-modifying repeat unit comprises the structure:

wherein:

n¹, n², m¹ and m² are independently 0-3, wherein n¹+m¹ and n²+m² areeach independently 2-3;

R¹⁶ and R¹⁷ are each independently selected from H, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group; and

each * is a site for covalent attachment to the backbone of thepolymeric dye.

-   Clause 11. The polymeric dye according to clause 5, wherein Z is a    covalent bond and the conjugation-modifying repeat unit comprises    the structure:

wherein:

R¹ to R⁴ are independently selected from hydrogen, alkyl, substitutedalkyl, halogen (e.g., fluoro), cyano,

with the proviso that R¹ and R² are not hydrogen;

each R⁵ is independently —(CH₂CH₂O)_(p)R′ wherein each p isindependently an integer from 0 to 50 (such as 6-50, 6-40, 6-30, 8-30,10-30 or 10-20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) andR′ is H, an alkyl or a substituted alkyl;

X is O or S; and

m is an integer from 0 to 10.

-   Clause 12. The polymeric dye according to any one of clauses 1-11,    wherein the polymeric dye comprises a plurality of π-conjugated    segments, each comprising one or more co-monomers selected from a    substituted or unsubstituted aryl or heteroaryl co-monomer, an    alkynyl co-monomer and a substituted or unsubstituted alkenyl    co-monomer.-   Clause 13. The polymeric dye according to clause 12, wherein the    polymeric dye comprises a plurality of π-conjugated segments, each    comprising one or more co-monomers selected from substituted or    unsubstituted poly(p-phenylene), substituted or unsubstituted    biphenyl, substituted or unsubstituted phenylene, substituted or    unsubstituted fluorene, substituted or unsubstituted carbazole,    substituted or unsubstituted silole, substituted or unsubstituted    benzooxazole, substituted or unsubstituted benzothiazole, alkynyl,    and substituted or unsubstituted alkenyl.-   Clause 14. The polymeric dye according to any one of clauses 1-13,    wherein the polymeric dye comprises a plurality of π-conjugated    segments, each comprising one or more co-monomers selected from the    following structures (a)-(k):

wherein:

X is O or S;

n is 1-6 (e.g., 1, 2 or 3)

R¹¹ is one or more optional substituents each independently selectedfrom hydrogen, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy,hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group; and

each * is a site for covalent attachment to the backbone of the polymer.

-   Clause 15. The polymeric dye according to clause 14, wherein:    structure (a) is selected from:

structure (g) is selected from:

structure (h) is selected from:

structure (i) is selected from:

wherein:

X is O or S;

Z¹ and Z² are independently selected from CR¹² and N;

Y³ is C or Si; and

each R¹¹ to R¹⁴ is independently hydrogen, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide,substituted sulfonamide, carboxy, carboxyamide, substituted carboxyamideand a water solubilizing group, and wherein two R¹⁴ groups areoptionally cyclically linked to form a fused carbocyclic or heterocyclicring (e.g., a partially saturated seven membered heterocyclic ring).

-   Clause 16. The polymeric dye according to clause 15, wherein each    R¹¹ to R¹⁴ is independently selected from H, F, methyl,    trifluoromethyl, cyano,

wherein:

X is O or S;

m is an integer from 0-10; and

R⁵ is —(CH₂CH₂O)_(p)R′ wherein R′ is H, alkyl or substituted alkyl(e.g., methyl) and each p is independently an integer from 0-40 (such as6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20).

-   Clause 17. The polymeric dye according to any one of clauses 1-16,    wherein the polymeric dye is of formula (I):

wherein:

each M¹ is independently selected from a substituted or unsubstitutedaryl co-monomer, a substituted or unsubstituted heteroaryl co-monomer,an alkynyl co-monomer and a substituted or unsubstituted alkenylco-monomer;

each M² is independently a conjugation-modifying repeat unit;

L¹ and L² are each independently a linker unit;

Z¹ is a linked chemoselective tag or a linked signaling chromophore(—Z¹);

Z² is a branched π-conjugated segment;

a-h are each independently 0 or 1, wherein a+b≥1 and b+e≥+1;

n is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250);

m and o are each independently from 0 to 10,000 (such as 0 to 1,000, 0to 500 or 0 to 250, e.g., 1 to 150, 2 to 250, 5 to 250 or 10 to 250);

p is from 1 to 100,000 (such as 1 to 10,000, 1 to 1,000, 1 to 500 or 1to 250, e.g., 2 to 250, 5 to 250 or 10 to 250); and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member.

-   Clause 18. The polymeric dye according to clause 17, wherein the    polymeric dye is of formula (II):

wherein:

M¹ and M³ are independently selected from an aryl co-monomer, aheteroaryl co-monomer, an alkynyl co-monomer and an alkenyl co-monomer;

Z is selected from a covalent bond, a saturated atom or group and afunctional group;

R¹¹ and R¹² are each independently selected from H, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group;

a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

n is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250);

m is from 0 to 10,000 (such as 0 to 1,000, 0 to 500 or 0 to 250, e.g., 1to 150, 2 to 250, 5 to 250 or 10 to 250);

p is from 1 to 100,000 (such as 1 to 10,000, 1 to 1,000, 1 to 500 or 1to 250, e.g., 2 to 250, 5 to 250 or 10 to 250); and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member.

-   Clause 19. The polymeric dye according to clause 17, wherein the    polymeric dye is of formula (III):

wherein:

M¹ is selected from an aryl co-monomer, a heteroaryl co-monomer, analkynyl co-monomer and an alkenyl co-monomer;

L¹ is a linker unit comprising an aryl co-monomer, a heteroarylco-monomer, an alkynyl co-monomer and an alkenyl co-monomer;

Z¹ is a linked chemoselective tag or a linked signaling chromophore(—Z¹);

Z is selected from a covalent bond, a saturated atom or group and afunctional group;

R¹¹ and R¹² are each independently selected from H, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group;

a, b, d and e are each independently 0 or 1, wherein a+b≥1 and d+e≥1;

n is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250);

m is from 0 to 10,000 (such as 0 to 1,000, 0 to 500 or 0 to 250, e.g., 1to 150, 2 to 250, 5 to 250 or 10 to 250);

p is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250); and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member.

-   Clause 20. The polymeric dye according to clause 17, wherein:

the polymeric dye is of formula (I) wherein a, b, g and h are each 1;

Z² has is a branched π-conjugated segment of formula (IV):

wherein M¹, M², L¹, Z¹ are as defined in clause 17; and

G³ is a terminal group, a π-conjugated segment, a linker or a linkedspecific binding member.

-   Clause 21. The polymeric dye according to clause 20, wherein:

the polymeric dye is of formula (I) wherein a, b, d, e, g, h are each 1;and

Z¹ is a linked functional signaling chromophore.

-   Clause 22. The polymeric dye according to any one of clauses 17-19,    wherein the polymeric dye is of formula (V):

wherein

Z is selected from a covalent bond, a saturated atom or group and afunctional group;

R¹-R³, R¹¹ and R¹² are each independently selected from H, alkyl,substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen,sulfonate, sulfonamide, substituted sulfonamide, carboxy, carboxyamide,substituted carboxyamide, a water solubilizing group, a linkedchemoselective tag and a linked signalling chromophore;

a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

n is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250);

m is from 0 to 10,000 (such as 0 to 1,000, 0 to 500 or 0 to 250, e.g., 1to 150, 2 to 250, 5 to 250 or 10 to 250);

p is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250); and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member.

-   Clause 23. The polymeric dye according to clause 22, wherein:

R¹-R³ are each independently a water solubilizing group; and

R² and R³ are the same.

-   Clause 24. The polymeric dye according to clause 22, wherein:

each R¹ and R² are each independently a water solubilizing group; and

R³ is a linked chemoselective tag or a linked signalling chromophore.

-   Clause 25. The polymeric dye according to any one of clauses 18-24,    wherein Z is selected from a saturated carbon atom, a saturated    heteroatom, a functional group (e.g., an oxygen and/or    nitrogen-containing functional group, such as a sulfonyl, a keto, an    amide, ester, carbonate, carbamate or urea) and a saturated linker.-   Clause 26. The polymeric dye according to clause 25, wherein:

Z is selected from —S—, —S(O)—, —SO₂—, —SO₂NR′—, —S(O)O—, —O—, —C(O)—,—C(O)NR′—, —C(O)O—, —OC(O)NR′—, —OC(O)O—, —NR′C(O)NR′—, —C(R⁵)(R⁶)—,—Si(R⁵)(R⁶)—, —N(R′)—, an alkyl linker (e.g., C₂-C₁₂, such as C₂-C₆substituted or unsubstituted alkyl linker) and a polyethylene glycollinker; and

each R′, R⁵ and R⁶ is independently selected from H, alkyl, substitutedalkyl, and wherein R⁵ and/or R⁶ is optionally cyclically linked to R¹²or R¹² to provide a benzo-fused carbocycle or heterocycle ring.

-   Clause 27. The polymeric dye according to clause 26, wherein Z is    selected from —S—, —S(O)—, —SO₂—, —O—, —C(O)—, —C(O)NR′—,    —OC(O)NR′—, —OC(O)O— and —NR′C(O)NR′—.-   Clause 28. The polymeric dye according to clause 27, wherein each Z    is —SO₂—.-   Clause 29. The polymeric dye according to clause 26, wherein Z is    C(R⁵)(R⁶)— or —Si(R⁵)(R⁶)—.-   Clause 30. The polymeric dye according to any one of clauses 22-29,    wherein each R¹ and R² are each independently a water solubilizing    group selected from:

wherein m is 0-10 (such as 1-6, e.g., 1, 2, 3, 4, 5 or 6) and each R⁵ isindependently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50(such as 6-50, 6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20) and R′ is H, an alkyl or a substitutedalkyl; and X is O.

-   Clause 31. The polymeric dye according to clause 22, wherein the    polymeric dye has the formula (VI):

wherein:

Dye is the linked signaling chromophore and L is a linker; WSG1 and WSG2are each independently a water solubilizing group (WSG) (e.g., asdescribed herein);

Z is selected from —S—, —S(O)—, —SO₂—, —SO₂NR′—, —S(O)O—, —O—, —C(O)—,—C(O)NR′—, —C(O)O—, —OC(O)NR′—, —OC(O)O—, —NR′C(O)NR′—, —C(R⁵)(R⁶)—,—Si(R⁵)(R⁶)—, —N(R′)—, an alkyl linker (e.g., C₂-C₁₂, such as C₂-C₆substituted or unsubstituted alkyl linker) and a polyethylene glycollinker;

each R′, R⁵ and R⁶ is independently selected from H, alkyl, substitutedalkyl, and wherein R⁵ and/or R⁶ is optionally cyclically linked to R¹²or R¹² to provide a benzo-fused carbocycle or heterocycle ring; and

x and y represent mol % values (e.g., y is 1-25 mol % and x is 99 mol %or less).

-   Clause 33. The polymeric dye according to clause 32, wherein Z is    selected from —S—, —S(O)—, —SO₂—, —O—, —C(O)—, —C(O)NR′—,    —OC(O)NR′—, —OC(O)O— and —NR′C(O)NR′—.-   Clause 34. The polymeric dye according to clause 32, wherein Z is    —SO₂—.-   Clause 35. The polymeric dye according to clause 32, wherein Z is    C(R⁵)(R⁶)— or —Si(R⁵)(R⁶)—.-   Clause 36. The polymeric dye according to any one of clauses 32-35,    wherein each WSG¹ and WSG² are each independently a water    solubilizing group selected from:

wherein m is 0-10 (such as 1-6, e.g., 1, 2, 3, 4, 5 or 6) and each R⁵ isindependently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50(such as 6-50, 6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20) and R′ is H, an alkyl or a substitutedalkyl; and X is O.

-   Clause 37. The polymeric dye according to clause 22, wherein the    polymeric dye has the formula (VII):

wherein:

R¹-R⁴ are each independently selected from H, alkyl, substituted alkyl,alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide, a water solubilizing group (WSG) (e.g., as describedherein), a linked chemoselective tag and a linked signallingchromophore;

a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

x and y represent mol % values (e.g., y is 1-25 mol % and x is 99 mol %or less);

p is from 1 to 10,000 (such as 1 to 1,000, 1 to 500 or 1 to 250, e.g., 2to 250, 5 to 250 or 10 to 250); and

G¹ and G² are independently selected from a terminal group, aπ-conjugated segment, a linker and a linked specific binding member.

-   Clause 38. The polymeric dye according to clause 37, wherein R¹-R³    are each independently a water solubilizing group; and R² and R³ are    the same.-   Clause 39. The polymeric dye according to clause 37, wherein the    subject polymeric dye has the formula (VIII):

wherein:

Dye is the linked signaling chromophore and L is a linker; each WSG1,each WSG2 and WSG3 are each independently a water solubilizing group(WSG) (e.g., as described herein); and x and y represent mol % values.In some cases, y is 1-25 mol % and x is 99 mol % or less.

-   Clause 40. The polymeric dye according to clause 39, wherein WSG1,    each WSG2 and WSG3 are independently a water solubilizing group    selected from:

wherein m is 0-10 (such as 1-6, e.g., 1, 2, 3, 4, 5 or 6) and each R⁵ isindependently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50(such as 6-50, 6-40, 6-30, 8-30, 10-30 or 10-20, e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20) and R′ is H, an alkyl or a substitutedalkyl; and X is O.

-   Clause 41. The polymeric dye according to any one of clauses 17-21,    wherein each M¹ and each M³ is independently selected from the    following structures (a)-(k):

wherein:

X is O or S;

n is 1-6 (e.g., 1, 2 or 3);

Y¹ is selected from —C(R¹¹)₂—, —Si(R¹¹)₂—, —NR¹¹—, —C(R¹¹)₂C(R¹¹)₂— and—C(R¹¹)₂C(R¹¹)₂—;

R¹¹ is one or more optional substituents each independently selectedfrom hydrogen, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy,hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate,sulfonamide, substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide and a water solubilizing group or wherein any two adjacentR¹¹ groups are optionally cyclically linked (e.g., to provide acarbocycle or heterocycle ring); and

each * is a site for covalent attachment to the backbone of the polymer.

-   Clause 42. The polymeric dye according to clause 41, wherein:-   structure (a) is selected from:

structure (g) is selected from:

structure (h) is selected from:

structure (i) is selected from:

wherein:

X is O or S;

Z¹ and Z² are independently selected from CR¹² and N;

Y³ is C or Si; and

each R¹¹ to R¹⁴ is independently hydrogen, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide and a water solubilizing group,and wherein two R¹⁴ groups are optionally cyclically linked to form afused carbocyclic or heterocyclic ring (e.g., a partially saturatedseven membered heterocyclic ring).

-   Clause 43. A polymeric tandem dye comprising:

a water solvated polymeric dye having a deep ultraviolet excitationspectrum and comprising a segment of π-conjugated co-monomers and aconjugation-modifying repeat unit; and

a signaling chromophore covalently linked to the water solvatedpolymeric dye in energy-receiving proximity therewith.

-   Clause 44. The polymeric tandem dye according to clause 43, wherein    the conjugation-modifying repeat unit comprises the structure:

wherein:

Z is selected from a covalent bond, a saturated atom, a saturated groupand a functional group, with the proviso that when Z is a covalent bondat least two of R²², R²³, R²⁵ and R²⁸ are not H (e.g., at least one ofR²² and R²³, and at least one of R²⁵ and R²⁸ is not H);

R²¹-R²⁸ are independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide, and a water solubilizing group,and wherein one or more of R²², R²³, R²⁵ and R²⁸ is optionallycyclically linked to Z to provide a benzo-fused carbocycle orheterocycle ring; and

each * is a site for covalent attachment to the backbone of thepolymeric dye.

-   Clause 45. The polymeric tandem dye according to clause 43, wherein    the signaling chromophore emission has a quantum yield of 0.1 or    more.-   Clause 46. The polymeric tandem dye according to clause 43, wherein    the polymeric dye has an extinction coefficient of 1×10⁶ M⁻¹ cm⁻¹ or    more.-   Clause 47. The polymeric tandem dye according to any one of clauses    43-46, wherein the multichromophore is substituted with non-ionic    side groups capable of imparting solubility in water in excess of 10    mg/mL.-   Clause 48. The polymeric tandem dye according to any one of clauses    43-47, wherein the signaling chromophore is a fluorophore.-   Clause 49. The polymeric tandem dye according to any one of clauses    43-47, wherein the signaling chromophore is a quencher.-   Clause 50. The polymeric tandem dye according to any one of clauses    43-49, wherein the signaling chromophore is selected from a    rhodamine, a coumarin, a xanthene, a cyanine, a polymethine, a    pyrene, a dipyrromethene borondifluoride, a napthalimide, a    phycobiliprotein, a peridinum chlorophyll protein, conjugates    thereof, and combinations thereof.-   Clause 51. The polymeric tandem dye according to any one of clauses    43-48 and 50, wherein the signaling chromophore is selected from    fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594,    tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,    carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,    coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight    350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight    633, DyLight 649, DyLight 680, DyLight 750, DyLight 800,    phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5,    JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimelhoxyfluorescein), NED, ROX    (5-(and -6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,    Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor®    350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa    Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633,    Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680,    7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2,    BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 576/589,    BODIPY® 581/591, BODIPY® 630/650, BODIPY® 650/665, BODIPY® R6G,    BODIPY® TMR, BODIPY® TR, conjugates thereof and combinations    thereof.-   Clause 52. The polymeric tandem dye according to any one of clauses    43-51, wherein:

the signaling chromophore is linked to a co-monomer comprising 1% to 20%by molarity of the multichromophore; and

the polymeric dye is a conjugated polymer comprising 5 or more repeatunits.

-   Clause 53. The polymeric tandem dye according to any one of clauses    43-48 and 50-52, wherein the signaling chromophore emission is    1.5-fold greater or more when excited by the polymeric dye as    compared to direct excitation of the acceptor chromophore with    incident light.-   Clause 54. The polymeric tandem dye according to any one of clauses    43-53, wherein the polymeric dye comprises a terminal group -L³-Z    where L³ is a linker and Z is a specific binding member.-   Clause 55. The polymeric tandem dye according to clause 54, wherein    the linker is selected from the group consisting of an alkyl, a    substituted alkyl, an alkyl-amido, an alkyl-amido-alkyl and a PEG    moiety.-   Clause 56. The polymeric tandem dye according to clause 55, wherein    -L³-Z is described by the following structure:

wherein: q is 0-12; and Z is the specific binding member.

-   Clause 57. The polymeric tandem dye according to any one of clauses    54-56, wherein Z is a biomolecule.-   Clause 58. The polymeric tandem dye according to clause 57, wherein    Z is an antibody.-   Clause 59. The polymeric tandem dye according to clause 57, wherein    Z is an antibody fragment or binding derivative thereof.-   Clause 60. The polymeric tandem dye according to clause 59, wherein    the antibody fragment or binding derivative thereof is selected from    the group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv,    a diabody and a triabody.-   Clause 61. The polymeric tandem dye according to any one of clauses    53-60, wherein the water solvated polymeric dye is a polymeric dye    according to any one of clauses 2-42.-   Clause 62. A labelled specific binding member, comprising:

a water solvated polymeric dye having a deep ultraviolet excitationspectrum and comprising a segment of π-conjugated co-monomers and aconjugation-modifying repeat unit; and

a specific binding member covalently linked to the polymeric dye.

-   Clause 63. The labelled specific binding member according to clause    62, wherein the conjugation-modifying repeat unit comprises the    structure:

wherein:

Z is selected from a covalent bond, a saturated atom, a saturated groupand a functional group, with the proviso that when Z is a covalent bondat least two of R²², R²³, R²⁵ and R²⁸ are not H (e.g., at least one ofR²² and R²³, and at least one of R²⁵ and R²⁸ is not H);

R²¹-R²⁸ are independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide, and a water solubilizing group,and wherein one or more of R²², R²³, R²⁵ and R²⁸ is optionallycyclically linked to Z to provide a benzo-fused carbocycle orheterocycle ring; and

each * is a site for covalent attachment to the backbone of thepolymeric dye.

-   Clause 64. The labelled specific binding member according to any one    of clauses 62-63, wherein the polymeric dye comprises a signaling    chromophore covalently linked to the polymeric dye in    energy-receiving proximity therewith.-   Clause 65. The labelled specific binding member according to any one    of clauses 62-64, wherein the specific binding member is an    antibody.-   Clause 66. The labelled specific binding member according to any one    of clauses 62-64, wherein the specific binding member is an antibody    fragment or binding derivative thereof.-   Clause 67. The labelled specific binding member according to clause    66, wherein the antibody fragment or binding derivative thereof is    selected from the group consisting of a Fab fragment, a F(ab′)₂    fragment, a scFv, a diabody and a triabody.-   Clause 68. The labelled specific binding member according to clause    64, wherein the signaling chromophore is selected from a rhodamine,    a coumarin, a xanthene, a cyanine, a polymethine, a pyrene, a    dipyrromethene borondifluoride, a napthalimide, a phycobiliprotein,    a peridinum chlorophyll protein, conjugates thereof, and    combinations thereof.-   Clause 69. The labelled specific binding member according to any one    of clauses 62-68, wherein the water solvated polymeric dye is a    polymeric dye according to any one of clauses 2-42.-   Clause 70. A method of evaluating a sample for the presence of a    target analyte, the method comprising:

(a) contacting the sample with a polymeric dye conjugate thatspecifically binds the target analyte to produce a labelling compositioncontacted sample, wherein the polymeric dye conjugate comprises:

-   -   (i) a polymeric dye having a deep ultraviolet excitation        spectrum according to any one of clauses 1-42; and    -   (ii) a specific binding member covalently linked to the        polymeric dye; and

(b) assaying the labelling composition contacted sample for the presenceof a polymeric dye conjugate-target analyte binding complex to evaluatewhether the target analyte is present in the sample.

-   Clause 71. The method according to clause 70, wherein the polymeric    dye comprises a signaling chromophore covalently linked to the    multichromophore in energy-receiving proximity therewith.-   Clause 72. The method according to any one of clauses 70-71, further    comprising contacting the sample with a second specific binding    member that is support bound and specifically binds the target    analyte.-   Clause 73. The method according to clause 72, wherein the support    comprises a magnetic particle.-   Clause 74. The method according to any one of clauses 70-73, wherein    the target analyte is associated with a cell.-   Clause 75. The method according to clause 75, wherein the target    analyte is a cell surface marker of the cell.-   Clause 76. The method according to clause 75, wherein the cell    surface marker is selected from the group consisting of a cell    receptor and a cell surface antigen-   Clause 77. The method according to clause 74, wherein the target    analyte is an intracellular target, and the method further comprises    lysing the cell.-   Clause 78. The method according to any one of clauses 70-77, wherein    the method further comprises flow cytometrically analyzing the    fluorescently labelled target analyte.-   Clause 79. A method of labelling a target molecule, the method    comprising:

contacting the target molecule with a water solvated polymeric dye toproduce a labelled target molecule, wherein the water solvated polymericdye has a deep ultraviolet excitation spectrum and comprises:

a segment of π-conjugated co-monomers;

a conjugation-modifying repeat unit; and

a conjugation tag that covalently links to the target molecule.

-   Clause 80. The method according to clause 79, wherein the    conjugation-modifying repeat unit comprises the structure:

wherein:

Z is selected from a covalent bond, a saturated atom, a saturated groupand a functional group, with the proviso that when Z is a covalent bondat least two of R²², R²³, R²⁵ and R²⁸ are not H (e.g., at least one ofR²² and R²³, and at least one of R²⁵ and R²⁸ is not H);

R²¹-R²⁸ are independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro orbromo), sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide, and a water solubilizing group,and wherein one or more of R²², R²³, R²⁵ and R²⁸ is optionallycyclically linked to Z to provide a benzo-fused carbocycle orheterocycle ring; and

each * is a site for covalent attachment to the backbone of thepolymeric dye.

-   Clause 81. The method according to any one of clauses 79-80, wherein    the polymeric dye comprises a signaling chromophore covalently    linked to the polymeric dye in energy-receiving proximity therewith.-   Clause 82. The method according to any one of clauses 79-81, further    comprising fluorescently detecting the labelled target molecule.-   Clause 83. The method according to any one of clauses 79-82, wherein    the conjugation tag comprises a terminal functional group selected    from an amino, a thiol, a hydroxyl, a hydrazine, a hydrazide, an    azide, an alkyne, maleimide, iodoacetyl, amine, an active ester and    a protein reactive group.-   Clause 84. The method according to any one of clauses 79-83, wherein    the target molecule is a specific binding member.-   Clause 85. The method according to clause 84, wherein the specific    binding member is an antibody.-   Clause 86. The method according to clause 85, wherein the specific    binding member is an antibody fragment or binding derivative    thereof.-   Clause 87. The method according to clause 86, wherein the antibody    fragment or binding derivative thereof is selected from the group    consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody    and a triabody.-   Clause 88. A flow cytometric system, comprising:

a flow cytometer comprising a flow path;

a composition in the flow path, wherein the composition comprises:

-   -   a sample; and    -   a labelled specific binding member comprising:        -   a water solvated polymeric dye having a deep ultraviolet            excitation spectrum according to any one of clauses 1-30;            and        -   a specific binding member that specifically binds a target            analyte and is covalently linked to the water solvated            polymeric dye.

-   Clause 89. The system according to clause 88, wherein the labelled    specific binding member further comprises a signaling chromophore    covalently linked to the polymeric dye in energy-receiving proximity    therewith.

-   Clause 90. The system according to any one of clauses 88-89, wherein    the composition further comprises a second specific binding member    that is support bound and specifically binds the target analyte.

-   Clause 91. The system according to clause 90, wherein the support    comprises a magnetic particle.

-   Clause 92. The system according to any one of clauses 88-91, wherein    the sample comprises a cell.

-   Clause 93. The system according to clause 92, wherein the target    analyte is a cell surface marker of the cell.

-   Clause 94. The system according to clause 93, wherein the cell    surface marker is selected from the group consisting of a cell    receptor and a cell surface antigen.

-   Clause 95. A kit comprising: a polymeric dye having a deep    ultraviolet excitation spectrum according to any one of clauses    1-42; and a container.

-   Clause 96. The kit according to clause 95, further comprising one or    more components selected from the group consisting of a polymeric    tandem dye, a fluorophore, a specific binding member, a specific    binding member conjugate, a cell, a support, a biocompatible aqueous    elution buffer and instructions for use.

-   Clause 97. The kit according to any one of clauses 95-96, wherein    the polymeric dye is covalently linked to a specific binding member.

-   Clause 98. The kit according to clause 97, wherein the specific    binding member is an antibody.

-   Clause 99. The kit according to clause 97, wherein the specific    binding member is an antibody fragment or binding derivative    thereof.

-   Clause 100. The kit according to clause 99, wherein the antibody    fragment or binding derivative thereof is selected from the group    consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody    and a triabody.

-   Clause 101. The kit according to any one of clauses 95-100, wherein    polymeric dye further comprises an acceptor signaling chromophore    covalently linked to the polymeric due in energy-receiving proximity    therewith.

EXAMPLES Example 1 Synthesis of Conjugated Polymer Via C—H BondArylation

The synthesis of the subject polymeric dyes can be achieved by atechnique called C—H bond arylation (Scheme 1). Other methods such as aSuzuki coupling method (Scheme 2) or a Stille coupling method (Scheme 3)can also be utilized. The following exemplary synthetic schemes can beadapted for the synthesis of the subject polymeric dyes having a varietyof co-monomers, as described herein.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

What is claimed is:
 1. A water solvated polymeric dye comprising: asegment of π-conjugated co-monomers; and a conjugation-modifying repeatunit; wherein the polymeric dye has a deep ultraviolet excitationspectrum, wherein the conjugation-modifying repeat unit comprises thestructure:

wherein: Z is selected from a covalent bond, a saturated atom, asaturated group and a functional group, with the proviso that when Z isa covalent bond at least two of R²², R²³, R²⁵ and R²⁸ are not H; R²¹—R²⁸are independently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide,substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide, and a water solubilizing group, and wherein one or more ofR²², R²³, R²⁵ and R²⁸ is optionally cyclically linked to Z to provide abenzo-fused carbocycle or heterocycle ring; and each * is a site forcovalent attachment to the backbone of the polymeric dye.
 2. Thepolymeric dye according to claim 1, wherein: a) Z is selected from asaturated carbon atom, a saturated heteroatom, a functional group and asaturated linker; b) Z is selected from —S—, —S(O)—, —SO₂—, —SO₂NR′—,—S(O)O—, —O—, —C(O)—, —C(O)NR′—, —C(O)O—, —OC(O)NR′—, —OC(O)O—,—NR′C(O)NR′—, —C(R⁵)(R⁶)—, —Si(R⁵)(R⁶)—, —N(R′)—, an alkyl linker and apolyethylene glycol linker; and each R′, R⁵ and R⁶ is independentlyselected from H, alkyl, substituted alkyl, or wherein R⁵ and/or R⁶ isoptionally cyclically linked to R²², R²³, R²⁵ or R²⁸ provide thebenzo-fused carbocycle or heterocycle ring; c) Z is selected from —S—,—S(O)—, —SO₂—, —O—, —C(O)—, —C(O)NR′—, —OC(O)NR′—, —OC(O)O— and—NR′C(O)NR′—; or d) Z is C(R⁵)(R⁶)— or —Si(R⁵)(R⁶)—.
 3. The polymericdye according to claim 2, wherein the conjugation-modifying repeat unitcomprises the structure:

wherein: n¹, n², m¹ and m² are independently 0-3, wherein n¹+m¹ andn²+m² are each independently 2-3; R¹⁶ and R¹⁷ are each independentlyselected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy,hydroxy, cyano, halogen, sulfonate, sulfonamide, substitutedsulfonamide, carboxy, carboxyamide, substituted carboxyamide and a watersolubilizing group; and each * is a site for covalent attachment to thebackbone of the polymeric dye.
 4. The polymeric dye according to claim2, wherein Z is a covalent bond and the conjugation-modifying repeatunit comprises the structure:

wherein: R¹ to R⁴ are independently selected from hydrogen, alkyl,substituted alkyl, halogen, cyano,

with the proviso that R¹ and R² are not hydrogen; each R⁵ isindependently —(CH₂CH₂O)_(p)R′ wherein p is an integer from 0 to 50 andR′ is H, an alkyl or a substituted alkyl; X is O or S; and m is aninteger from 0 to
 10. 5. The polymeric dye according to claim 1, whereinthe polymeric dye comprises a plurality of π-conjugated segments, eachcomprising one or more co-monomers selected from: a) a substituted orunsubstituted aryl or heteroaryl co-monomer, an alkynyl co-monomer and asubstituted or unsubstituted alkenyl co-monomer; b) substituted orunsubstituted poly(p-phenylene), substituted or unsubstituted biphenyl,substituted or unsubstituted phenylene, substituted or unsubstitutedfluorene, substituted or unsubstituted carbazole, substituted orunsubstituted silole, substituted or unsubstituted benzooxazole,substituted or unsubstituted benzothiazole, alkynyl, and substituted orunsubstituted alkenyl; c) the following structures (a)-(k):

wherein: X is O or S; n is 1-6; R¹¹ is one or more optional substituentseach independently selected from hydrogen, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide,substituted sulfonamide, carboxy, carboxyamide, substituted carboxyamideand a water solubilizing group; and each * is a site for covalentattachment to the backbone of the polymer.
 6. The polymeric dyeaccording to claim 1, wherein the polymeric dye is of formula (I):

wherein: each M¹ is independently selected from a substituted orunsubstituted aryl co-monomer, a substituted or unsubstituted heteroarylco-monomer, an alkynyl co-monomer and a substituted or unsubstitutedalkenyl co-monomer; each M² is independently a conjugation-modifyingrepeat unit; L¹ and L² are each independently a linker unit; Z¹ is alinked chemoselective tag or a linked signaling chromophore (—Z¹); Z² isa branched π-conjugated segment; a-h are each independently 0 or 1,wherein a+b≥1 and b+e+h≥1; n is from 1 to 250; m and o are eachindependently from 0 to 250; p is from 1 to 100,000; and G¹ and G² areindependently selected from a terminal group, a π-conjugated segment, alinker and a linked specific binding member.
 7. The polymeric dyeaccording to claim 6, wherein: a) the polymeric dye is of formula (II):

wherein: M¹ and M³ are independently selected from an aryl co-monomer, aheteroaryl co-monomer, an alkynyl co-monomer and an alkenyl co-monomer;Z is selected from a covalent bond, a saturated atom or group and afunctional group; R¹¹ and R¹² are each independently selected from H,alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano,halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide and a water solubilizing group;a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1; nis from 1 to 250; m is from 0 to 250; p is from 1 to 100,000; and G¹ andG² are independently selected from a terminal group, a π-conjugatedsegment, a linker and a linked specific binding member; or b) thepolymeric dye is of formula (III):

wherein: M¹ is selected from an aryl co-monomer, a heteroarylco-monomer, an alkynyl co-monomer and an alkenyl co-monomer; L¹ is alinker unit comprising an aryl co-monomer, a heteroaryl co-monomer, analkynyl co-monomer and an alkenyl co-monomer; Z¹ is a linkedchemoselective tag or a linked signaling chromophore (—Z¹); Z isselected from a covalent bond, a saturated atom or group and afunctional group; R¹¹ and R¹² are each independently selected from H,alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano,halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxy,carboxyamide, substituted carboxyamide and a water solubilizing group;a, b, d and e are each independently 0 or 1, wherein a+b≥1 and d+e≥1; nis from 1 to 250; m is from 0 to 250; p is from 1 to 10,000; and G¹ andG² are independently selected from a terminal group, a π-conjugatedsegment, a linker and a linked specific binding member.
 8. The polymericdye according to claim 6, wherein: the polymeric dye is of formula (I)wherein a, b, g and h are each 1; Z² has is a branched π-conjugatedsegment of formula (IV):

G³ is a terminal group, a π-conjugated segment, a linker or a linkedspecific binding member.
 9. The polymeric dye according to claim 6,wherein the polymeric dye is of formula (V):

wherein Z is selected from a covalent bond, a saturated atom or groupand a functional group; R¹-R³, R¹¹ and R¹² are each independentlyselected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy,hydroxy, cyano, halogen, sulfonate, sulfonamide, substitutedsulfonamide, carboxy, carboxyamide, substituted carboxyamide, a watersolubilizing group, a linked chemoselective tag and a linked signallingchromophore; a, b, c and e are each independently 0 or 1, wherein a+b≥1and c+d≥1; n is from 1 to 250; m is from 0 to 250; p is from 1 to10,000; and G¹ and G² are independently selected from a terminal group,a π-conjugated segment, a linker and a linked specific binding member.10. The polymeric dye according to claim 9, wherein: a) R¹-R³ are eachindependently a water solubilizing group; and R² and R³ are the same; orb) R¹ and R² are each independently a water solubilizing group; and R³is a linked chemoselective tag or a linked signalling chromophore. 11.The polymeric dye according to claim 6, wherein: a) Z is selected from asaturated carbon atom, a saturated heteroatom, a functional group and asaturated linker; b) Z is selected from —S—, —S(O)—, —SO₂—, —SO₂NR′—,—S(O)O—, —O—, —C(O)—, —C(O)NR′—, —C(O)O—, —OC(O)NR′—, —OC(O)O—,—NR′C(O)NR′—, —C(R⁵)(R⁶)—, —Si(R⁵)(R⁶)—, —N(R′)—, an alkyl linker (e.g.,C₂-C₁₂, such as C₂-C₆ substituted or unsubstituted alkyl linker) and apolyethylene glycol linker; and each R′, R⁵ and R⁶ is independentlyselected from H, alkyl, substituted alkyl, and wherein R⁵ and/or R⁶ isoptionally cyclically linked to R¹² or R¹² to provide a benzo-fusedcarbocycle or heterocycle ring; c) Z is selected from —S—, —S(O)—,—SO₂—, —O—, —C(O)—, —C(O)NR′—, —OC(O)NR′—, —OC(O)O— and —NR′C(O)NR′—; ord) Z is C(R⁵)(R⁶)— or —Si(R⁵)(R⁶)—.
 12. A polymeric tandem dyecomprising: a water solvated polymeric dye having a deep ultravioletexcitation spectrum and comprising a segment of π-conjugated co-monomersand a conjugation-modifying repeat unit; and a signaling chromophorecovalently linked to the water solvated polymeric dye inenergy-receiving proximity therewith, wherein the conjugation-modifyingrepeat unit comprises the structure:

wherein: Z is selected from a covalent bond, a saturated atom, asaturated group and a functional group, with the proviso that when Z isa covalent bond at least two of R²², R²³, R²⁵ and R²⁸ are not H; R²¹-R²⁸are independently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide,substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide, and a water solubilizing group, and wherein one or more ofR²², R²³, R²⁵ and R²⁸ is optionally cyclically linked to Z to provide abenzo-fused carbocycle or heterocycle ring; and each * is a site forcovalent attachment to the backbone of the polymeric dye.
 13. Thepolymeric tandem dye according to claim 12, wherein the signalingchromophore is: a) a fluorophore; b) a quencher; c) selected from arhodamine, a coumarin, a xanthene, a cyanine, a polymethine, a pyrene, adipyrromethene borondifluoride, a napthalimide, a phycobiliprotein, aperidinum chlorophyll protein, conjugates thereof, and combinationsthereof; or d) a fluorescent dye.
 14. The polymeric tandem dye accordingto claim 12, wherein: the signaling chromophore is linked to aco-monomer comprising 1% to 20% by molarity of the multichromophore; andthe polymeric dye is a conjugated polymer comprising 5 or more repeatunits.
 15. A labelled specific binding member, comprising: a watersolvated polymeric dye having a deep ultraviolet excitation spectrum andcomprising a segment of π-conjugated co-monomers and aconjugation-modifying repeat unit; and a specific binding membercovalently linked to the polymeric dye, wherein theconjugation-modifying repeat unit comprises the structure:

wherein: Z is selected from a covalent bond, a saturated atom, asaturated group and a functional group, with the proviso that when Z isa covalent bond at least two of R²², R²³, R²⁵ and R²⁸ are not H; R²¹-R²⁸are independently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide,substituted sulfonamide, carboxy, carboxyamide, substitutedcarboxyamide, and a water solubilizing group, and wherein one or more ofR²², R²³, R²⁵ and R²⁸ is optionally cyclically linked to Z to provide abenzo-fused carbocycle or heterocycle ring; and each * is a site forcovalent attachment to the backbone of the polymeric dye.
 16. Thelabelled specific binding member according to claim 15, wherein thepolymeric dye comprises a signaling chromophore covalently linked to thepolymeric dye in energy-receiving proximity therewith.
 17. The labelledspecific binding member according to claim 15, wherein: a) the specificbinding member is an antibody; or b) the specific binding member is anantibody fragment or binding derivative thereof.